Chemical Synthesis of Interleukin-6 for Mirror-Image Screening.

Interleukin-6 (IL-6), a multifunctional cytokine, is an attractive therapeutic target for immunological and inflammatory diseases. We investigated the chemical synthesis of IL-6 and its enantiomer (d-IL-6) using a sequential N-to-C native chemical ligation strategy from six peptide segments. Solubilizing Trt-K10 tags improved the intermediate solubility and served as protecting groups during the metal-free desulfurization to facilitate the synthesis of full-length IL-6 protein. Synthetic l-IL-6 and recombinant IL-6 exhibited nearly identical structural and binding properties. The symmetrical binding property of d-IL-6 was also demonstrated by functional analysis using IL-6-binding peptides. The resulting functional d-IL-6 was employed to screen a phage-displayed antibody fragment library, leading to the identification of several d-IL-6-binding single-domain antibodies. This work will contribute to the development of novel, potent IL-6 inhibitors without the adverse effects of undesired immune activation.

Construction of the Akuammiline Alkaloid Core Structure via Stereoselective E-Ring Formation.

Construction of the core structure of akuammiline alkaloids with three-dimensional cage-like structures for their diversity-oriented synthesis was investigated. Extensive exploration centered around the introduction of nitrogen functional groups and construction of the E-ring in an intramolecular or intermolecular manner revealed that a Claisen rearrangement approach involving intramolecular amination provided a common precursor, potentially facilitating divergent access to various types of akuammiline alkaloids.

Synthesis of Sulfilimines via Visible-Light-Mediated Triplet Energy Transfer to Sulfonyl Azides.

Sulfilimines and their derivatives have garnered considerable interest in both synthetic and medicinal chemistry. Photochemical nitrene transfer to sulfides is known as a conventional synthetic approach to sulfilimines. However, the existing methods have a limited substrate scope stemming from the incompatibility of singlet nitrene intermediates with nucleophilic functional groups. Herein, we report the synthesis of N-sulfonyl sulfilimines via visible-light-mediated energy transfer to sulfonyl azides, uncovering the previously overlooked reactivity of triplet nitrenes with sulfides. This reaction features broad functional group tolerance, water compatibility, and amenability to the late-stage functionalization of drugs. Thus, this work represents an important example of energy transfer chemistry that overcomes challenges in traditional synthetic methods.

Mirror-Image Human Serum Albumin Domain III as a Tool for Analyzing Site II-Dependent Molecular Recognition.

Human serum albumin (HSA) as a drug carrier can significantly improve the pharmacokinetic profiles of short-lived therapeutics. Conjugation of albumin-binding moieties (ABMs) to therapeutic agents may prolong their serum half-life by promoting their association with endogenous HSA. To discover a new molecular class of ABMs from mirror-image chemical space, a preparation protocol for bioactive HSA domain III and its d-enantiomer (d-HSA domain III) was established. Structural and functional analyses suggested that the synthetic protein enantiomers exhibited mirror-image structures and stereoselective neonatal fragement crystallizable receptor (FcRn) recognition. Additionally, the ligand-binding properties of synthetic l-HSA domain III were comparable with those of site II in native HSA, as confirmed using site II-selective fluorescent probes and an esterase substrate. Synthetic d-HSA domain III is an attractive tool for analyzing the site II-dependent molecular recognition properties of HSA.

Engineering a Low-Immunogenic Mirror-Image VHH against Vascular Endothelial Growth Factor.

Immunogenicity is a major caveat of protein therapeutics. In particular, the long-term administration of protein therapeutic agents leads to the generation of antidrug antibodies (ADAs), which reduce drug efficacy while eliciting adverse events. One promising solution to this issue is the use of mirror-image proteins consisting of d-amino acids, which are resistant to proteolytic degradation in immune cells. We have recently reported the chemical synthesis of the enantiomeric form of the variable domain of the antibody heavy chain (d-VHH). However, identifying mirror-image antibodies capable of binding to natural ligands remains challenging. In this study, we developed a novel screening platform to identify a d-VHH specific for vascular endothelial growth factor A (VEGF-A). We performed mirror-image screening of two newly constructed synthetic VHH libraries displayed on T7 phage and identified VHH sequences that effectively bound to the mirror-image VEGF-A target (d-VEGF-A). We subsequently synthesized a d-VHH candidate that preferentially bound the native VEGF-A (l-VEGF-A) with submicromolar affinity. Furthermore, immunization studies in mice demonstrated that this d-VHH elicited no ADAs, unlike its corresponding l-VHH. Our findings highlight the utility of this novel d-VHH screening platform in the development of protein therapeutics exhibiting both reduced immunogenicity and improved efficacy.

Structure-activity relationship studies of tetrahydroquinolone derivatives as GPR41 modulators.

GPR41, a G protein-coupled receptor, serves as a sensor for short-chain fatty acids and plays a crucial role in regulating multiple physiological processes such as the maintenance of metabolic and immune homeostasis. Therefore, the modulation of GPR41 has garnered attention as a potential strategy for the treatment of various disorders. We conducted a structure-activity relationship study on a lead tetrahydroquinolone derivative bearing a 2-(trifluoromethoxy)benzene group that displayed antagonistic activity toward GPR41. Modification of the aryl group attached to the furan moiety revealed that derivatives containing di- or trifluorobenzene, instead of 2-(trifluoromethoxy)benzene, exhibited agonistic activity toward GPR41, comparable with the reported agonistic modulator AR420626. These results suggest that the aryl group plays a pivotal role in regulating the activity of compounds toward GPR41, providing valuable insights for the design of GPR41 modulators.

Synthesis of the full-length hepatitis B virus core protein and its capsid formation.

Chronic infection with hepatitis B virus (HBV) is a major cause of cirrhosis and liver cancer. Capsid assembly modulators can induce error-prone assembly of HBV core proteins to prevent the formation of infectious virions, representing promising candidates for treating chronic HBV infections. To explore novel capsid assembly modulators from unexplored mirror-image libraries of natural products, we have investigated the synthetic process of the HBV core protein for preparing the mirror-image target protein. In this report, the chemical synthesis of full-length HBV core protein (Cp183) containing an arginine-rich nucleic acid-binding domain at the C-terminus is presented. Sequential ligations using four peptide segments enabled the synthesis of Cp183 via convergent and C-to-N direction approaches. After refolding under appropriate conditions, followed by the addition of nucleic acid, the synthetic Cp183 assembled into capsid-like particles.

Synthesis of labionin and avionin precursors via a nitrogen-centred-radical-triggered 1,5-HAT reaction of Tris derivatives.

Labionin and avionin are non-proteinogenic amino acids containing 2,4-diamino-2-(mercaptomethyl)pentanedioic acid that forms the core structures of spirocyclic peptides including labyrinthopeptin A2 and microvionin, respectively. We have developed a diastereoselective synthetic route to labionin and avionin precursors. This route highlights the formation of the quaternary carbon stereocenter of an α,α-disubstituted amino acid via a regioselective 1,5-HAT reaction of a Tris derivative.

Synthetic studies on the extracellular domain of the T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) using Trt-K10 solubilizing tags.

The T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is an inhibitory immunoreceptor expressed on lymphocytes that serves as a promising target for cancer immunotherapy. In this study, facile synthetic protocols to produce the extracellular domain of TIGIT were investigated for applications of TIGIT in mirror-image screening. During the synthesis via sequential native chemical ligations, we encountered problems with significantly poor solubility of the ligated products. Introducing trityl-type solubilizing auxiliaries, which also functioned as temporary protecting groups for cysteine residues, facilitated a flexible order of ligations and efficient purification protocols. After refolding under appropriate conditions, the synthetic TIGIT showed a sufficient affinity toward its target ligand CD155.

The identification of novel small extracellular vesicle (sEV) production modulators using luciferase-based sEV quantification method.

Small extracellular vesicles (sEVs) are nano-sized vesicles secreted from various cells that contain bioactive metabolites and function as key regulators for intercellular communication. sEVs modulate diverse biological and pathological processes in the body, and the amount of circulating sEVs has been reported to correlate with certain disease progression. Therefore, the identification of small molecular compounds that can control sEV production may become a novel therapeutic strategy. In this study, a rapid, highly sensitive sEV quantification method utilizing fusion proteins consisting of Gaussia luciferase (gLuc) reporter protein and sEV markers (CD63 and CD82) was developed. A total of 480 compounds were screened to identify potent inducers and inhibitors of gLuc activity. Two novel compounds, KPYC08425 and KPYC12163, showed significant and dose-dependent changes in gLuc activity with minimal cytotoxicity based on the LDH assay. The efficacy of these two compounds was further evaluated by protein quantification of the isolated sEVs. Further evaluation of KPYC12163 suggested that the autolysosomal pathway may be involved in its inhibitory effect on sEV production.