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.

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.

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 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.

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.

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.

Gold(I)-Catalyzed Cascade Cyclization of Alkynyl Indoles for the Stereoselective Construction of the Quaternary Carbon Center of Akuammiline Alkaloids.

A gold-catalyzed cyclization reaction of alkynyl-indoles has been developed for the stereoselective construction of the quaternary carbon center of fused indolines. This reaction efficiently produces fused indolines via diastereoselective 6-endo-dig cyclization controlled by a bulky TIPS group, followed by nucleophilic attack of the carboxy group on the resulting imine. The lactone moiety of the fused indoline can be reductively cleaved to produce a tricyclic indoline, which could be useful for the synthesis of akuammiline alkaloids.

Design and Synthesis of Monobody Variants with Low Immunogenicity.

Mirror-image proteins (d-proteins) are promising scaffolds for drug discovery because of their high proteolytic stability and low immunogenic properties. Facile and reproducible processes for the preparation of functional d-proteins are required for their application in therapeutic biologics. In this study, we designed and synthesized a novel monobody variant with two cysteine substitutions that facilitate the synthetic process via sequential native chemical ligations and improve protein stability by disulfide bond formation. The synthetic anti-GFP monobody in this model study exhibited good binding affinity to the target enhanced green fluorescent protein. In vivo administration of the synthetic anti-GFP monobody (l-monobody) to mice induced antidrug antibody (ADA) production, whereas no ADA production was observed following immunization with the mirror-image anti-GFP monobody (d-monobody). These results suggest that the synthetic d-monobody is a non-antibody protein scaffold with low immunogenic properties.

Mirror-Image Single-Domain Antibody for a Novel Nonimmunogenic Drug Scaffold.

Immunogenic responses by protein therapeutics often lead to reduced therapeutic effects and/or adverse effects via the generation of neutralizing antibodies and/or antidrug antibodies (ADA). Mirror-image proteins of the variable domain of the heavy chain of the heavy chain antibody (VHH) are potential novel protein therapeutics with high-affinity binding to target proteins and reduced immunogenicity because these mirror-image VHHs (d-VHHs) are less susceptible to proteolytic degradation in antigen-presenting cells (APCs). In this study, we investigated the preparation protocols of d-VHHs and their biological properties, including stereoselective target binding and immunogenicity. Initially, we established a facile synthetic process of two model VHHs [anti-GFP VHH and PMP12A2h1 (monomeric VHH of caplacizumab)] and their mirror-image proteins by three-step native chemical ligations (NCLs) from four peptide segments. The folded synthetic VHHs (l-anti-GFP VHH and l-PMP12A2h1) bound to the target proteins (EGFP and vWF-A1 domain, respectively), while their mirror-image proteins (d-anti-GFP VHH and d-PMP12A2h1) showed no binding to the native proteins. For biodistribution studies, l-VHH and d-VHH with single radioactive indium diethylenetriamine-pentaacid (111In-DTPA) labeling at the C-terminus were designed and synthesized by the established protocol. The distribution profiles were essentially similar between l-VHH and d-VHH, in which the probes accumulated in the kidney within 15 min after intravenous administration in mice, because of the small molecular size of VHHs. Comparative assessment of the immunogenicity responses revealed that d-VHH-induced levels of ADA generation were significantly lower than those of native VHH, regardless of the peptide sequences and administration routes. The resulting scaffold investigated should be applicable in the design of d-VHHs with various C-terminal CDR3 sequences, which can be identified by screening using display technologies.

Design of Synthetic Surrogates for the Macrolactone Linker Motif in Coibamide A.

A marine cyanobacterial cyclic depsipeptide, coibamide A (CbA), inhibits the mammalian protein secretory pathway by blocking the Sec61 translocon, which is an emerging drug target for cancer and other chronic diseases. In our previous structure-activity relationship study of CbA, the macrolactone ester linker was replaced with alkyl/alkenyl surrogates to provide synthetically accessible macrocyclic scaffolds. To optimize the cellular bioactivity profile of CbA analogues, novel lysine mimetics having ß- and ε-methyl groups have now been designed and synthesized by a stereoselective route. A significant increase in cytotoxicity was observed upon introduction of these two methyl groups, corresponding to the d-MeAla α-methyl and MeThr ß-methyl of CbA. All synthetic products retained the ability to inhibit secretion of a model Sec61 substrate. Tandem evaluation of secretory function inhibition in living cells and cytotoxicity was an effective strategy to assess the impact of structural modifications to the linker for ring closure.

Development of a 1:1-binding biparatopic anti-TNFR2 antagonist by reducing signaling activity through epitope selection.

Conventional bivalent antibodies against cell surface receptors often initiate unwanted signal transduction by crosslinking two antigen molecules. Biparatopic antibodies (BpAbs) bind to two different epitopes on the same antigen, thus altering crosslinking ability. In this study, we develop BpAbs against tumor necrosis factor receptor 2 (TNFR2), which is an attractive immune checkpoint target. Using different pairs of antibody variable regions specific to topographically distinct TNFR2 epitopes, we successfully regulate the size of BpAb-TNFR2 immunocomplexes to result in controlled agonistic activities. Our series of results indicate that the relative positions of the two epitopes recognized by the BpAb are critical for controlling its signaling activity. One particular antagonist, Bp109-92, binds TNFR2 in a 1:1 manner without unwanted signal transduction, and its structural basis is determined using cryo-electron microscopy. This antagonist suppresses the proliferation of regulatory T cells expressing TNFR2. Therefore, the BpAb format would be useful in designing specific and distinct antibody functions.

Archaeal Glycerolipids Are Recognized by C-Type Lectin Receptor Mincle.

Recently, various metabolites derived from host microbes have been reported to modulate the immune system, with potential involvement in health or diseases. Archaea, prokaryotic organisms, are present in the human body, but their connection with the host is largely unknown when compared to other microorganisms such as bacteria. This study focused on unique glycerolipids from symbiotic methanogenic archaea and evaluated their activities toward an innate immune receptor. The results revealed that archaeal lipids were recognized by the C-type lectin receptor Mincle and induced immune responses. A concurrent structure-activity relationship study identified the key structural features of archaeal lipids required for recognition by Mincle. Subsequent gene expression profiling suggested qualitative differences between the symbiotic archaeal lipid and the pathogenic bacteria-derived lipid. These findings have broad implications for understanding the function of symbiotic archaea in host health and diseases.

Establishment of an MR1 Presentation Reporter Screening System and Identification of Phenylpropanoid Derivatives as MR1 Ligands.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that are modulated by ligands presented on MHC class I-related proteins (MR1). These cells have attracted attention as potential drug targets because of their involvement in the initial response to infection and various disorders. Herein, we have established the MR1 presentation reporter assay system employing split-luciferase, which enables the efficient exploration of MR1 ligands. Using our screening system, we identified phenylpropanoid derivatives as MR1 ligands, including coniferyl aldehyde, which have an ability to inhibit the MR1-MAIT cell axis. Further, the structure-activity relationship study of coniferyl aldehyde analogs revealed the key structural features of ligands required for MR1 recognition. These results will contribute to identifying a broad range of endogenous and exogenous MR1 ligands and to developing novel MAIT cell modulators.

Spontaneous Cervical Epidural Hematoma Presenting with Respiratory Failure: A Case Report and Literature Review.

CASE: A 62-year-old woman who had an unremarkable medical history presented with sudden headache and neck pain. After the presentation, complete quadriplegia and respiratory arrest developed, and the patient was urgently intubated. Magnetic resonance imaging revealed an extensive epidural hematoma (EH), and emergency hematoma evacuation was performed. At the 1-year follow-up visit, the patient had no motor deficits. CONCLUSION: We reported a case of spontaneous cervical EH presenting with respiratory failure that was successfully treated with surgical management. Literature review has shown that the surgical outcome is very poor; nevertheless, prompt surgical decompression of the spinal cord can minimize neurological sequelae.

Gold(I)-Catalyzed Bis-Cyclization of Allenynes for the Synthesis of Strained and Planar Polycyclic Compounds.

A gold-catalyzed cascade cyclization of naphthalene-tethered allenynes gave strained fused phenanthrene derivatives. The reaction proceeds through the nucleophilic reaction of an alkyne with the activated allene to generate a vinyl cation intermediate, followed by arylation with a tethered naphthalene ring to form the 4H-cyclopenta[def]phenanthrene (CPP) scaffold. When using aryl-substituted substrates on the alkyne terminus, the gold-catalyzed reaction produced dibenzofluorene derivatives along with the CPP derivatives. Selective formation of CPP and dibenzofluorene derivatives depending on the reaction conditions is also presented.

A Proximity-Induced Fluorogenic Reaction Triggered by Antibody-Antigen Interactions with Adjacent Epitopes.

Proximity-induced chemical reactions are site-specific and rapid by taking advantage of their high affinity and highly selective interactions with the template. However, reactions induced solely by antibody-antigen interactions have not been developed. Herein, we propose a biepitopic antigen-templated chemical reaction (BATER) as a novel template reaction. In BATER, reactive functional groups are conjugated to two antibodies that interact with two epitopes of the same antigen to accelerate the reaction. We developed a method for visualizing the progress of BATER using fluorogenic click chemistry for optimal antibody selection and linker design. The reaction is accelerated in the presence of a specific antigen in a linker length-dependent manner. The choice of the antibody epitope is important for a rapid reaction. This design will lead to various applications of BATER in living systems.

Construction of a Bicyclo[2.2.2]octene Skeleton via a Visible-Light-Mediated Radical Cascade Reaction of Amino Acid Derivatives with N-(2-Phenyl)benzoyl Groups.

Bridged polycyclic ring systems constitute the core structures of numerous natural products and biologically active molecules. We found that simple biphenyl substrates derived from amino acids participate in a radical cascade reaction under visible light irradiation in the presence of [Ir{dF(CF3)ppy}2(dtbpy)]PF6 to enable the direct construction of bicyclo[2.2.2]octene structures. Isotopic labeling experiments suggested that intramolecular hydrogen atom transfer is involved in the cascade processes.

Discovery of Bis-sulfonamides as Novel Inhibitors of Mitochondrial NADH-Quinone Oxidoreductase (Complex I).

Mitochondrial oxidative phosphorylation (OXPHOS) is an essential cellular metabolic process that generates ATP. The enzymes involved in OXPHOS are considered to be promising druggable targets. Through screening of an in-house synthetic library with bovine heart submitochondrial particles, we identified a unique symmetric bis-sulfonamide, KPYC01112 (1) as an inhibitor targeting NADH-quinone oxidoreductase (complex I). Structural modifications of KPYC01112 (1) led to the discovery of the more potent inhibitors 32 and 35 possessing long alkyl chains (IC50 = 0.017 and 0.014 µM, respectively). A photoaffinity labeling experiment using a newly synthesized photoreactive bis-sulfonamide ([125I]-43) revealed that it binds to the 49-kDa, PSST, and ND1 subunits which make up the quinone-accessing cavity of complex I.

Host metabolic benefits of prebiotic exopolysaccharides produced by Leuconostoc mesenteroides.

Fermented foods demonstrate remarkable health benefits owing to probiotic bacteria or microproducts produced via bacterial fermentation. Fermented foods are produced by the fermentative action of several lactic acid bacteria, including Leuconostoc mesenteroides; however, the exact mechanism of action of these foods remains unclear. Here, we observed that prebiotics associated with L. mesenteroides-produced exopolysaccharides (EPS) demonstrate substantial host metabolic benefits. L. mesenteroides-produced EPS is an indigestible α-glucan, and intake of the purified form of EPS improved glucose metabolism and energy homeostasis through EPS-derived gut microbial short-chain fatty acids, and changed gut microbial composition. Our findings reveal an important mechanism that accounts for the effects of diet, prebiotics, and probiotics on energy homeostasis and suggests an approach for preventing lifestyle-related diseases by targeting bacterial EPS.

Gold(I)-Catalyzed Benzylic C(sp3 )-H Functionalizations: Divergent Synthesis of Indole[a]- and [b]-Fused Polycycles.

Phenyl azides substituted by an (alkylphenyl)ethynyl group facilitate benzylic sp3 (C-H) functionalization in the presence of a JohnPhosAu catalyst, resulting in indole-fused tetra- and pentacycles via divergent N- or C-cyclization. The chemoselectivity is influenced depending on the counter-anion, the electron density of the α-imino gold(I) carbene, and the alkyl groups stabilizing the benzylic carbocation originating from a 1,5-hydride shift. An isotopic labeling experiment demonstrates the involvement of an indolylgold(I) species resulting from a tautomerization that is much faster than the deauration. The formation of a benzylic sp3 (C-H) functionalization leading to an indole-fused seven-membered ring is also demonstrated.