The Application of Microsampling Disks in Circular Dichroism Spectroscopy for Peptide and Nucleic Acid Drugs.

In recent years, there has been a growing focus on the development of medium-sized drugs based on peptides or nucleic acids owing to their potential therapeutic benefits. As some of these medium-sized drugs exert their therapeutic effects by adopting specific secondary structures, evaluating their conformational states is crucial to ensure the efficacy, quality, and safety of the drug products. It is important to assess the structural integrity of biomolecular therapeutics to guarantee their intended pharmacological activity and maintain the required standards for drug development and manufacturing. One widely utilized technique for quality evaluation is secondary structural analysis using circular dichroism (CD) spectroscopy. Given the higher production and quality control costs associated with medium-sized drugs compared with small-molecule drugs, developing analytical techniques that enable CD analysis with reduced sample volumes is highly desirable. Herein, we focused on a microsampling disk-type cell as a potential solution for reducing the required sample volume. We investigated whether CD spectral analysis using a microsampling disk could provide equivalent spectra compared with the standard cell (sample volume: approx. 300 µL). Our findings demonstrated that the microsampling disk (sample volume: 2-10 µL) could be successfully applied to CD spectral analysis of peptide and nucleic acid drugs, paving the way for more efficient and cost-effective quality evaluation processes.

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 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 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.

Synthesis of Norgestomet and its 17ß-isomer and evaluation of their agonistic activities against progesterone receptor.

Norgestomet is a synthetic progesterone derivative applied in veterinary medicine to control estrus and ovulation in cattle. Norgestomet has been widely used in the livestock industry to promote the synchronization of estrus in cattle and increase pregnancy rates. However, highly reproducible synthetic methods for Norgestomet have been rarely reported. Here, we described a method for the synthesis of Norgestomet and performed quantitative NMR analysis to determine the purity of the products. Moreover, the agonistic activity of the synthesized compounds against progesterone receptors (PRs) was evaluated using an alkaline phosphatase assay. We synthesized Norgestomet with 97.9% purity that exhibited agonistic activity against PR with EC50 values of 4.5 nM. We also synthesized the 17ß-isomer of Norgestomet with 92.7% purity that did not exhibit any PR agonistic activity. The proposed synthetic route of Norgestomet can facilitate the assessment of residual Norgestomet in foods.

Structure-activity relationship study of amphipathic antimicrobial peptides using helix-destabilizing sarcosine.

Antimicrobial peptides (AMPs) are potential therapeutic agents against bacteria. We recently showed that a rationally designed AMP, termed Stripe, with an amphipathic distribution of native cationic and hydrophobic amino acids on its helical structure exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria with negligible hemolytic activity and cytotoxicity. In this study, the structure-activity relationship of Stripe was elucidated by designing a series of antimicrobial peptides whereby amino acid residues of Stripe were exchanged with helix-destabilizing sarcosine residues. Stripe 1-5 peptides with hydrophobic amino acids substituted with sarcosine were predominantly unstructured and showed no antimicrobial activity, except against Escherichia coli (E. coli) (DH5α) cells. The activity against E. coli (DH5α) cells and the helicity of Stripe 1-5 peptides decreased concomitantly as the number of sarcosine residue substitutions increased. Stripe 1-5 peptides showed no hemolytic activity or cytotoxicity. The results indicate that sarcosine substitutions provide an approach to study the structure-activity relationship of helical AMPs, and the helicity of Stripe is an important feature defining its activity.

Development of Antimicrobial Stapled Peptides Based on Magainin 2 Sequence.

Magainin 2 (Mag2), which was isolated from the skin of the African clawed frog, is a representative antimicrobial peptide (AMP) that exerts antimicrobial activity via microbial membrane disruption. It has been reported that the helicity and amphipathicity of Mag2 play important roles in its antimicrobial activity. We investigated and recently reported that 17 amino acid residues of Mag2 are required for its antimicrobial activity, and accordingly developed antimicrobial foldamers containing α,α-disubstituted amino acid residues. In this study, we further designed and synthesized a set of Mag2 derivatives bearing the hydrocarbon stapling side chain for helix stabilization. The preferred secondary structures, antimicrobial activities, and cell-membrane disruption activities of the synthesized peptides were evaluated. Our analyses revealed that hydrocarbon stapling strongly stabilized the helical structure of the peptides and enhanced their antimicrobial activity. Moreover, peptide 2 stapling between the first and fifth position from the N-terminus showed higher antimicrobial activity than that of Mag2 against both gram-positive and gram-negative bacteria without exerting significant hemolytic activity. To investigate the modes of action of tested peptides 2 and 8 in antimicrobial and hemolytic activity, electrophysiological measurements were performed.

De Novo Design of Cell-Penetrating Foldamers.

Cell-penetrating peptides (CPPs) have gained much attention as carriers of hydrophilic molecules, such as drugs, peptides, and nucleic acids, into cells. CPPs are mainly composed of cationic amino acid residues, which play an important role in their intracellular uptake via interactions with acidic groups on cell surfaces. In addition, the secondary structures of CPPs also affect their cell-membrane permeability. Based on this knowledge, a variety of cell-penetrating foldamers (oligomers that form organized secondary structures) have been developed to date. In this account, we describe recent attempts to develop cell-penetrating foldamers containing various building blocks, and their application as DDS carriers.

Development of Photoswitchable Estrogen Receptor Ligands.

Photopharmacology has attracted attention as an approach for the development of novel therapeutics because it allows regulation of the bioactivity of compounds based on their conformational change by photo-irradiation. Previously, we have reported several types of selective estrogen receptor (ER) modulators based on diphenylmethane skeleton. To develop novel photopharmacological reagents, we designed and synthesized a set of ER ligands based on azobenzene skeleton, which can switch its conformation following UV irradiation. Our results showed that after UV irradiation, the Z-form of the synthesized compound 9 interacted with ERα, with a KD value of 2.5 µM, whereas the E-form of compound 9 did not bind ability to ERα at 10 µM.

Inhibition of ß-amyloid-induced neurotoxicity by planar analogues of procyanidin B3.

Reactive oxygen species (ROS) are known to be produced during the amyloid beta (Aß) aggregation process. Both ROS production and Aß fibril formation can result in nerve cell injury. Proanthocyanidins are oligomers of catechin that can act as inhibitors of Aß aggregation. Procyanidin B3 (Cat-Cat), the dimer of (+)-catechin, can easily cross the blood-brain barrier. Previously, we synthesized two derivatives of Cat-Cat, namely Cat-PCat and PCat-PCat, in which the geometry of one or both catechin molecules in Cat-Cat was constrained to be planar. The antioxidative activities of Cat-PCat and PCat-PCat were found to be stronger than that of Cat-Cat, with PCat-PC at exhibiting the most potent activity. These compounds are predicted to protect against Aß-induced neurotoxicity via inhibition of Aß aggregation as well as by antioxidative effects toward Aß-induced intracellular ROS generation. PCat-PCat exhibited the most potent neuroprotective effects against Aß-induced cytotoxicity, which resulted from inhibition of ß-sheet structure formation during the Aß aggregation process. PCat-PCat may be a promising lead compound for the treatment of Alzheimer's disease.

Design and synthesis of estrogen receptor ligands with a 4-heterocycle-4-phenylheptane skeleton.

The estrogen receptor (ER), a member of the nuclear receptor (NR) family, is involved in the regulation of physiological effects such as reproduction and bone homeostasis. Approximately 70% of human breast cancers are hormone-dependent and ERα-positive, and, thus, ER antagonists are broadly used in breast cancer therapy. We herein designed and synthesized a set of ER antagonists with a 4-heterocycle-4-phenylheptane skeleton.

Structural development of non-secosteroidal vitamin D receptor (VDR) ligands without any asymmetric carbon.

Non-secosteroidal VDR ligands without any assymmetric carbon were designed and synthesized based on the structure of the previously reported non-secosteroidal VDR agonist LG190178. The VDR-agonistic activity of all synthesized compounds was evaluated, and 7b emerged as a potent agonist activity with an EC50 value of 9.26 nM. Moreover, a docking simulation analysis was also performed to determine the binding mode of 7b with VDR-LBD.

Structural Development of Cell-Penetrating Peptides Containing Cationic Proline Derivatives.

We designed and synthesized a series of cell-penetrating peptides containing cationic proline derivatives (ProGu) that exhibited responsive changes in their secondary structures to the cellular environment. Effects of the peptide length and steric arrangement of the side chain in cationic proline derivatives [Pro4SGu and Pro4RGu] on their secondary structures and cell membrane permeability were investigated. Moreover, peptides 3 and 8 exhibited efficient intracellular delivery of plasmid DNA.

Rational Design and Synthesis of Post-Functionalizable Peptide Foldamers as Helical Templates.

In this study, we developed post-functionalizable helical peptides composed of Leu, Aib, and Azl residues. We show that the synthesized peptides 1 and 2 form helical structures, and may be modified using specific side chain or several functional groups by the click reaction without influencing their secondary structures.

Preorganized Cyclic α,α-Disubstituted α-Amino Acids Bearing Functionalized Side Chains That Act as Peptide-Helix Inducers.

Preorganized cyclic α,α-disubstituted α-amino acids (dAA) bearing functionalized side chains that acted as peptide-helix inducers, which could be used for solid-phase peptide synthesis, were designed and synthesized. Furthermore, a helical octapeptide with the following amino acid sequence was prepared, and its preferred conformation was analyzed based on its CD spectra: Ac-X1EYSAX2KA-NH2 (11: X1 = ApiC4N3, X2 = Ac6c). The side-chain azido functional group of peptide 11 was efficiently converted to various 1,2,3-triazole groups via Huisgen 1,3-dipolar cycloaddition reactions involving different types of alkynes. The new cyclic dAA derivatives, which combine the advantages of conformational preorganization and side-chain functional groups, should prove to be a useful tool for the further development of biologically active peptides.

Development of a peptide-based inducer of protein degradation targeting NOTCH1.

We previously developed a protein knockdown system by small-molecule hybrid compounds named SNIPERs (Specific and Nongenetic IAP-dependent Protein Erasers). Here we report a peptide-based protein knockdown system for inducing degradation of a transcriptional factor NOTCH1. The molecules designed were composed of two biologically active scaffolds: a peptide that binds to the surface of the target protein NOTCH1 and a small-molecule MV1 that binds to the E3 ubiquitin ligase inhibitor of apoptosis protein (IAP), which are expected to cross-link these proteins in cells. Hybrid molecules specifically induced the degradation of the NOTCH1 protein by the proteasome. This system could be a useful method to develop various degradation inducers against a large number of proteins to which small-molecule ligands have not been found.

Development of an ON/OFF switchable fluorescent probe targeting His tag fused proteins in living cells.

The fluorescent labeling of target proteins is useful for analyzing their functions and localization in cells, and several fluorescent probes have been developed. However, the fusion of tags such as green fluorescent protein (GFP) to target proteins occasionally affects their functions and/or localization in living cells. Therefore, an imaging method that uses short peptide tags such as hexa-histidine (the His tag) has been attracting increasing attention. Few studies have investigated ON/OFF switchable fluorescent probes for intracellular His-tagged proteins. We herein developed a novel ON/OFF switchable probe for imaging targeted intracellular proteins fused with a CH6 tag, which is composed of one cysteine residue and six histidine residues.

Efficient synthesis of a multi-substituted diphenylmethane skeleton as a steroid mimetic.

Steroids are important components of cell membranes and are involved in several physiological functions. A diphenylmethane (DPM) skeleton has recently been suggested to act as a mimetic of the steroid skeleton. However, difficulties are associated with efficiently introducing different substituents between two phenyl rings of the DPM skeleton, and, thus, further structural development based on the DPM skeleton has been limited. We herein developed an efficient synthetic method for introducing different substituents into two phenyl rings of the DPM skeleton. We also synthesized DPM-based estrogen receptor (ER) modulators using our synthetic method and evaluated their ER transcriptional activities.

Development of helix-stabilized antimicrobial peptides composed of lysine and hydrophobic α,α-disubstituted α-amino acid residues.

Lysine-based amphipathic nonapeptides, including homochiral peptides [Ac-(l-Lys-l-Lys-Xaa)3-NH2 (Xaa=Gly, Ala, Aib, Ac5c, or Ac6c) and Ac-(d-Lys-d-Lys-Aib)3-NH2], a heterochiral peptide [Ac-(l-Lys-d-Lys-Aib)3-NH2], and a racemic mixture of diastereomeric peptides [Ac-(rac-Lys-rac-Lys-Aib)3-NH2] were designed and synthesized to investigate the relationship between their preferred secondary structures and their antimicrobial activity. Peptide 5, [Ac-(l-Lys-l-Lys-Ac6c)3-NH2] formed a stable α-helical structure and exhibited strong activity against Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa).