The ovomucoid third domain from silver pheasant (OMSVP3), a typical Kazal-type inhibitor, strongly inhibits different serine proteases of various specificities, i.e., chymotrypsin, Streptomyces griseus protease, subtilisin, and elastase. Structural studies have suggested that conformational flexibility in the reactive site loop of the free inhibitor may be related to broad specificity of the ovomucoid. On the basis of the structural homology between OMSVP3 and ascidian trypsin inhibitor (ATI), which has a cystine-stabilized alpha-helical (CSH) motif in the sequence, we prepared the disulfide variant of OMSVP3, introducing an engineered disulfide bond between positions 14 and 39 near the reactive site (Met18-Glu19) by site-directed mutagenesis. The disulfide variant P14C/N39C retained potent inhibitory activities toward alpha-chymotrypsin (CHT) and S. griseus proteases A and B (SGPA and SGPB), while this variant lost most of its inhibitory activity toward porcine pancreatic elastase (PPE). We determined the solution structure of P14C/N39C, as well as that of wild-type OMSVP3, by two-dimensional nuclear magnetic resonance (2D NMR) methods and compared their structures to elucidate the structural basis of the inhibitory specificity change. For the molecular core consisting of a central alpha-helix and a three-stranded antiparallel beta-sheet, essentially no structural difference was detected between the two (pairwise rmsd value = 0.41 A). In contrast to this, a significant difference was detected in the loop from Cys8 to Thr17, where in P14C/N39C it has drawn approximately 4 A nearer the central helix to form the engineered Cys14-Cys39 bond. Concomitantly, the Tyr11-Pro12 cis-peptide linkage, which is highly conserved in ovomucoid third domains, was isomerized to the trans configuration. Such structural change in the loop near the reactive site may possibly affect the inhibitory specificity of P14C/N39C for the corresponding proteases.

We report a potent cationic lipid, SST-02 ((3-hydroxylpropyl)dilinoleylamine), which possesses a simple chemical structure and is synthesized just in one step. Cationic lipids are key components of siRNA-lipid nanoparticles (LNP), which may serve as potential therapeutic agents for various diseases. For a decade, chemists have given enhanced potency and new functions to cationic lipids along with structural complexity. In this study, we conducted a medicinal chemistry campaign pursuing chemical simplicity and found that even dilinoleylmethylamine (SST-01) and methylpalmitoleylamine could be used for the in vitro and in vivo siRNA delivery. Further optimization revealed that a hydroxyl group boosted potency, and SST-02 showed an ID 50 of 0.02 mg/kg in the factor VII (FVII) model. Rats administered with 3 mg/kg of SST-02 LNP did not show changes in body weight, blood chemistry, or hematological parameters, while the AST level decreased at a dose of 5 mg/kg. The use of SST-02 avoids a lengthy synthetic route and may thus decrease the future cost of nucleic acid therapeutics.

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Nucleic acid medicine is the next-generation therapeutic modality for refractory diseases with its unique mode of action as an alternative to traditional therapies. A nucleic acid delivery system targeted to liver was validated clinically; however, the delivery system of nucleic acids targeting solid tumors following systemic administration is not efficient enough for clinical use. In this study, we first utilized an antisense oligonucleotide (ASO) and polyethylene glycol (PEG) in one-to-one conjugation (PEG-ASO) at the endo-position of the ASO (endo-PEG-ASO). The effects of ASO modification position, PEG structure and molecular weight, and PEG-ASO tumor accumulation were evaluated in vivo. The endo-PEG-ASO showed prolonged pharmacokinetics and enhanced tumor accumulation compared with the conventional ASO and the PEG-ASO modified at the ASO exo-position (exo-PEG-ASO), indicating that the modification position of PEG is crucial for targeting tumors. We also observed that the endo-PEG-ASO indicated possibility of enhanced permeability inside the tumor. Further research is needed to optimize the linker in the endo-PEG-ASO for clinical application as a novel and promising therapeutic format for targeting solid tumors.

siRNAs are being developed as a novel therapeutic modality; however, problems impeding their application in extrahepatic tissues persist, including inadequate stability in biological environments and inefficient drug delivery system to target tissues. Thus, technological improvements that enable robust silencing of target messenger RNA (mRNA) in extrahepatic tissues are necessary. We developed prodrug type covalently closed siRNA (circular siRNA) as a novel nucleic acid agent to knockdown target genes in extrahepatic tissues by systemic administration without drug delivery components. Circular siRNA, which is chemically synthesizable, can assume optimal structures for efficient knockdown using its cleavable linker; namely, circular and linear structure in extracellular and intracellular environment, respectively. In this study, we investigated circular siRNA physicochemical properties, knockdown mechanism, and characteristics in vitro, as well as pharmacokinetics, accumulation, knockdown activity, and safety in vivo. Our circular siRNA exhibited higher stability against serum and exonucleases, increased cellular uptake, and stronger knockdown activity without transfection reagent in vitro than linear siRNA. Furthermore, after systemic administration to mice, circular siRNA showed prolonged circulation and improved knockdown activity in the liver, kidney, and muscle, without causing adverse effects. Circular siRNA may represent an additional platform for RNAi therapeutics, providing alternate solutions for disease treatment.