Development of an Anti-HER2 Single-Chain Variable Antibody Fragment Construct for High-Yield Soluble Expression in Escherichia coli and One-Step Chromatographic Purification.

Although single-chain variable fragment (scFv) is recognized as a highly versatile scaffold of recombinant antibody fragment molecules, its overexpression in Escherichia coli often leads to the formation of inclusion bodies. To address this issue, we devised and tested four different constructs, named v21, v22, v23 and v24, for producing anti-human epidermal growth factor receptor 2 (HER2) scFv. Among them, the v24 construct obtained from N-terminal fusion of maltose-binding protein (MBP) and subsequent tobacco etch virus protease (TEV) was identified as the most efficient construct for the production of anti-HER2 scFv. Aided by an MBP tag, high-yield soluble expression was ensured and soluble scFv was liberated in cells via autonomous proteolytic cleavage by endogenously expressed TEV. The isolated scFv containing a C-terminal hexahistidine tag was purified through a one-step purification via nickel-affinity chromatography. The purified scFv exhibited a strong (nanomolar Kd) affinity to HER2 both in vitro and in cells. Structural and functional stabilities of the scFv during storage for more than one month were also assured. Given the great utility of anti-HER2 scFv as a basic platform for developing therapeutic and diagnostic agents for cancers, the v24 construct and methods presented in this study are expected to provide a better manufacturing system for producing anti-HER2 scFv with various industrial applications.

Doxorubicin covalently conjugated heparin displays anti-cancer activity as a self-assembled nanoparticle with a low-anticoagulant effect.

Heparin is a glycosaminoglycans (GAGs) member and well-known FDA-approved anticoagulant that has been widely used in the clinic for 100 years. It has also been evaluated in various fields for further clinical applications, such as in anti-cancer or anti-inflammatory therapy beyond its anticoagulant effect. Here, we sought to utilize heparin molecules as drug carriers by directly conjugating the anticancer drug doxorubicin to the carboxyl group of unfractionated heparin. Given the molecular action of doxorubicin in intercalating DNA, it is expected to be less effective when structurally combined with other molecules. However, by utilizing doxorubicin molecules to produce reactive oxygen species (ROS), we found that the heparin-doxorubicin conjugates have significant cytotoxic ability to kill CT26 tumor cells with low anticoagulant activity. Several doxorubicin molecules were bound to heparin to provide sufficient cytotoxic capability and self-assembly ability due to their amphiphilic properties. The self-assembled formation of these nanoparticles was demonstrated through DLS, SEM and TEM. The cytotoxic ROS-generating doxorubicin-conjugated heparins could inhibit tumor growth and metastasis in CT26-bearing Balb/c animal models. Our results demonstrate that this cytotoxic doxorubicin-based heparin conjugate can significantly inhibit tumor growth and metastasis, thus showing promise as a potential new anti-cancer therapeutic.

Development of a Peptide-Based Nano-Sized Cathepsin B Inhibitor for Anticancer Therapy.

Numerous cathepsin B inhibitors have been developed and are under investigation as potential cancer treatments. They have been evaluated for their ability to inhibit cathepsin B activity and reduce tumor growth. However, they have shown critical limitations, including low anticancer efficacy and high toxicity, due to their low selectivity and delivery problems. In this study, we developed a novel peptide and drug conjugate (PDC)-based cathepsin B inhibitor using cathepsin-B-specific peptide (RR) and bile acid (BA). Interestingly, this RR and BA conjugate (RR-BA) was able to self-assemble in an aqueous solution, and as a result, it formed stable nanoparticles. The nano-sized RR-BA conjugate showed significant cathepsin B inhibitory effects and anticancer effects against mouse colorectal cancer (CT26) cells. Its therapeutic effect and low toxicity were also confirmed in CT26 tumor-bearing mice after intravenous injection. Therefore, based on these results, the RR-BA conjugate could be developed as an effective anticancer drug candidate for inhibiting cathepsin B in anticancer therapy.

Novel Activity of ODZ10117, a STAT3 Inhibitor, for Regulation of NLRP3 Inflammasome Activation.

The NLRP3 inflammasome serves as a host defense mechanism against various pathogens, but there is growing evidence linking its activation in sterile condition to diverse inflammatory diseases. Therefore, the identification of specific inhibitors that target NLRP3 inflammasome activation is meaningful and important for novel therapies for NLRP3 inflammasome-associated diseases. In this study, we identified a chemical compound, namely ODZ10117 (ODZ), that showed NLRP3 inflammasome-targeting anti-inflammatory effects during the screening of a chemical library for anti-inflammatory activity. Although ODZ was initially discovered as a STAT3 inhibitor, here we found it also has inhibitory activity on NLRP3 inflammasome activation. ODZ inhibited the cleavage of caspase-1 and IL-1ß-induced canonical NLRP3 inflammasome triggers, but had no effect on those induced by AIM2 or NLRC4 triggers. Mechanistically, ODZ impairs NLRP3 inflammasome activation through the inhibition of NLRP3-NEK7 interaction that is required for inflammasome formation. Moreover, the results obtained from the in silico docking experiment suggested that ODZ targets NLRP3 protein, which provides evidence for the specificity of ODZ to the NLRP3 inflammasome. Furthermore, ODZ administration significantly reduced MSU-induced IL-1ß release and the mortality rate of mice with LPS-induced sepsis. Collectively, these results demonstrate a novel effect of ODZ10117 in regulating NLRP3 inflammasome activation both in vitro and in vivo, making it a promising candidate for the treatment of NLRP3-inflammasome-associated immune disorders and cancer.

Cornus officinalis Seed Extract Inhibits AIM2-Inflammasome Activation and Attenuates Imiquimod-Induced Psoriasis-like Skin Inflammation.

The AIM2 inflammasome is an innate immune system component that defends against cytosolic bacteria and DNA viruses, but its aberrant activation can lead to the progression of various inflammatory diseases, including psoriasis. However, there have been few reports of specific inhibitors of AIM2 inflammasome activation. In this study, we aimed to investigate the inhibitory activity of ethanolic extracts of seeds of Cornus officinalis (CO), a herb and food plant used in traditional medicine, on AIM2-inflammasome activation. We found that CO inhibited the release of IL-1ß induced by dsDNA in both BMDMs and HaCaT cells, but that it showed no effect on the release of IL-1ß induced by NLRP3 inflammasome triggers, such as nigericin and silica, or the NLRC4 inflammasome trigger flagellin. Furthermore, we demonstrated that CO inhibited the cleavage of caspase-1, an inflammasome activation marker, and an upstream event, the translocation and speck formation of ASC. In addition, further experiments and mechanistic investigations revealed that CO can inhibit AIM2 speck formation induced by dsDNA in AIM2-overexpressing HEK293T cells. To verify the correlation in vivo, we investigated the efficacy of CO in an imiquimod (IMQ)-induced psoriasis model, which has reported associations with the AIM2 inflammasome. We found that topical application of CO alleviated psoriasis-like symptoms, such as erythema, scaling, and epidermal thickening, in a dose-dependent manner. Moreover, CO also significantly decreased IMQ-induced expression of AIM2 inflammasome components, including AIM2, ASC, and caspase-1, and led to the elevation of serum IL-17A. In conclusion, our results suggest that CO may be a valuable candidate for the discovery of AIM2 inhibitors and the regulation of AIM2-related diseases.

Polyphenols Coordinated with Cu (II) in an Aqueous System Build Ion-Channel Coatings on Hair Surfaces.

Recently, developments in the field of cosmetics have led to a renewed interest in hair dyeing. However, damage to the hair during the dyeing process has increased hesitation in attempting hair dyeing. As a result, hair dyes with minimal side effects have been in constant demand, and are being developed. In this study, natural-extract polyphenols, pyrogallol, and gallic acid are coordinated by CuCl2 in a NaCl aqueous solution to form an oligomer, which creates an ion-channel coating on the hair surface to protect it. This work attempts to develop fast, simple, and damage-free hair-dye ingredients based on pyrogallol and gallic acid. The morphology and elements of polyphenols coated on hair are characterized. The results reveal that the hair is dyed with the polyphenol-based dye reagent successfully. Moreover, the thickness of the dyed hair continuously rises ten times after dyeing. The tensile strength of the dyed hair is also measured, showing an upward and downward trend. These results reflect the fact that pyrogallol and gallic acid are considered to be the essential and functional polyphenols, and can build ion blocks on hair, which can create new multifunctional coating materials.

10-mer and 9-mer WALK Peptides with Both Antibacterial and Anti-Inflammatory Activities.

Natural antimicrobial peptides (AMPs) are multifunctional host defense peptides (HDPs) that are valuable for various therapeutic applications. In particular, natural and artificial AMPs with dual antibacterial immunomodulatory functions emerged as promising candidates for the development of therapeutic agents to treat infectious inflammation. In an effort to develop useful AMP variants with short lengths and simple amino acid composition, we devised a de novo design strategy to generate a series of model peptide isomer sequences, named WALK peptides, i.e., tryptophan (W)-containing amphipathic-helical (A) leucine (L)/lysine (K) peptides. Here, we generated two groups of WALK peptide isomers: W2L4K4 (WALK244.01~WALK244.10) and W2L4K3 (WALK243.01~WALK243.09). Most showed apparent antibacterial activities against both Gram-positive and Gram-negative bacteria at a concentration of approximately 4 µg/mL along with varied hemolytic activities against human red blood cells. In addition, some exhibited significant anti-inflammatory activities without any significant cytotoxicity in macrophages. Collectively, these results suggest that the two selected peptides, WALK244.04 and WALK243.04, showed promise for the development of antibacterial and anti-inflammatory agents.

In Vitro and In Vivo Model for Sepsis Through Non-canonical NLRP3 Inflammasome Activation.

Sepsis is a complex disorder related to dysregulation of the host response to infection and is a major health problem worldwide owing to its high mortality rates. However, the exact mechanisms causing sepsis remains unclear because of the complexity of the underlying pathways. Dysregulation of non-canonical NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation induces septic shock by promoting pyroptosis and pro-inflammatory cytokine production (e.g., interleukin-1ß) via caspase-11 and Gasdermin-D. Herein, we describe a rapid, simple method for evaluation of the degree of sepsis by investigating non-canonical inflammasome activation in both in vitro and in vivo models. The method is expected to be useful for testing and screening drugs for the treatment of sepsis.

Heparin and Its Derivatives: Challenges and Advances in Therapeutic Biomolecules.

Heparin has been extensively studied as a safe medicine and biomolecule over the past few decades. Heparin derivatives, including low-molecular-weight heparins (LMWH) and heparin pentasaccharide, are effective anticoagulants currently used in clinical settings. They have also been studied as functional biomolecules or biomaterials for various therapeutic uses to treat diseases. Heparin, which has a similar molecular structure to heparan sulfate, can be used as a remarkable biomedicine due to its uniquely high safety and biocompatibility. In particular, it has recently drawn attention for use in drug-delivery systems, biomaterial-based tissue engineering, nanoformulations, and new drug-development systems through molecular formulas. A variety of new heparin-based biomolecules and conjugates have been developed in recent years and are currently being evaluated for use in clinical applications. This article reviews heparin derivatives recently studied in the field of drug development for the treatment of various diseases.

NLRP3 Ubiquitination-A New Approach to Target NLRP3 Inflammasome Activation.

In response to diverse pathogenic and danger signals, the cytosolic activation of the NLRP3 (NOD-, LRR-, and pyrin domain-containing (3)) inflammasome complex is a critical event in the maturation and release of some inflammatory cytokines in the state of an inflammatory response. After activation of the NLRP3 inflammasome, a series of cellular events occurs, including caspase 1-mediated proteolytic cleavage and maturation of the IL-1ß and IL-18, followed by pyroptotic cell death. Therefore, the NLRP3 inflammasome has become a prime target for the resolution of many inflammatory disorders. Since NLRP3 inflammasome activation can be triggered by a wide range of stimuli and the activation process occurs in a complex, it is difficult to target the NLRP3 inflammasome. During the activation process, various post-translational modifications (PTM) of the NLRP3 protein are required to form a complex with other components. The regulation of ubiquitination and deubiquitination of NLRP3 has emerged as a potential therapeutic target for NLRP3 inflammasome-associated inflammatory disorders. In this review, we discuss the ubiquitination and deubiquitination system for NLRP3 inflammasome activation and the inhibitors that can be used as potential therapeutic agents to modulate the activation of the NLRP3 inflammasome.

Mitigating Effect of Lindera obtusiloba Blume Extract on Neuroinflammation in Microglial Cells and Scopolamine-Induced Amnesia in Mice.

Lindera obtusiloba Blume (family, Lauraceae), native to Northeast Asia, has been used traditionally in the treatment of trauma and neuralgia. In this study, we investigated the neuroinflammatory effect of methanol extract of L. obtusiloba stem (LOS-ME) in a scopolamine-induced amnesia model and lipopolysaccharide (LPS)-stimulated BV2 microglia cells. LOS-ME downregulated the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, inflammatory cytokines, and inhibited the phosphorylation of nuclear factor kappa-B (NF-ĸB) and extracellular signal-regulated kinase (ERK) in LPS-stimulated BV2 cells. Male C57/BL6 mice were orally administered 20 and 200 mg/kg of LOS-ME for one week, and 2 mg/kg of scopolamine was administered intraperitoneally on the 8th day. In vivo behavioral experiments (Y-maze and Morris water maze test) confirmed that LOS-ME alleviated cognitive impairments induced by scopolamine and the amount of iNOS expression decreased in the hippocampus of the mouse brain. Microglial hyper-activation was also reduced by LOS-ME pretreatment. These findings suggest that LOS-ME might have potential in the treatment for cognitive improvement by regulating neuroinflammation.

The Role of Tumor Necrosis Factor Alpha (TNF-α) in Autoimmune Disease and Current TNF-α Inhibitors in Therapeutics.

Tumor necrosis factor alpha (TNF-α) was initially recognized as a factor that causes the necrosis of tumors, but it has been recently identified to have additional important functions as a pathological component of autoimmune diseases. TNF-α binds to two different receptors, which initiate signal transduction pathways. These pathways lead to various cellular responses, including cell survival, differentiation, and proliferation. However, the inappropriate or excessive activation of TNF-α signaling is associated with chronic inflammation and can eventually lead to the development of pathological complications such as autoimmune diseases. Understanding of the TNF-α signaling mechanism has been expanded and applied for the treatment of immune diseases, which has resulted in the development of effective therapeutic tools, including TNF-α inhibitors. Currently, clinically approved TNF-α inhibitors have shown noticeable potency in a variety of autoimmune diseases, and novel TNF-α signaling inhibitors are being clinically evaluated. In this review, we briefly introduce the impact of TNF-α signaling on autoimmune diseases and its inhibitors, which are used as therapeutic agents against autoimmune diseases.

Down-regulation of pro-necroptotic molecules blunts necroptosis during myogenesis.

Cell death and differentiation are closely related at the molecular level. Differentiation of skeletal muscle cells attenuates susceptibility to apoptosis. Necroptosis has recently been recognized as a form of regulated cell death but its role in myogenesis has not been studied. This study aimed to compare the sensitivity to TNF-induced necroptosis in skeletal muscle at the undifferentiated (myoblasts) and differentiated (myotubes) stages. Surprisingly, our results showed that TNF-induced necroptosis was blunted during myoblast differentiation. Moreover, our data revealed that the key molecules involved in necroptosis, including receptor-interacting serine/threonine protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL), were significantly down-regulated during myogenic differentiation, resulting in suppression of necroptosis signal transduction in differentiated myotubes. In addition, RIPK1, RIPK3, and MLKL expression levels were significantly lower in the skeletal muscle of adult mice than in newborn mice, suggesting that the susceptibility to necroptosis might be attenuated in differentiated muscle tissue. In conclusion, this study revealed that expression of key molecules involved in necroptosis is down-regulated during muscle differentiation, which results in the differentiation of muscles becoming insensitive to necroptotic cell death.

Regulation of Caspase-8 Activity at the Crossroads of Pro-Inflammation and Anti-Inflammation.

Caspase-8 has been classified as an apoptotic caspase, and its initial definition was an initiator of extrinsic cell death. During the past decade, the concept of caspase-8 functioning has been changed by findings of its additional roles in diverse biological processes. Although caspase-8 was not originally thought to be involved in the inflammation process, many recent works have determined that caspase-8 plays an important role in the regulatory functions of inflammatory processes. In this review, we describe the recent advances in knowledge regarding the manner in which caspase-8 modulates the inflammatory responses concerning inflammasome activation, cell death, and cytokine induction.

Immunostimulatory Activity of Polysaccharides Extracted from Celosia cristata Flowers.

Macrophages play a major role in innate immune responses by producing a variety of immune mediators and cytokines. The stimulation of macrophages by natural products may lead to an enhanced innate immune system. This study evaluated the immunostimulatory effects of a polysaccharide-rich crude fraction of Celosia cristata L. flowers (CCP) on murine macrophages. CCP treatment induced the production of inducible nitric oxide synthase, cyclooxygenase-2, and cytokines by macrophages. Mechanistically, the activation of mitogen-activated protein kinases, NF-κB and toll-like receptor 4 were found to be associated with the stimulatory functions of CCP. CCP was found to be primarily composed of galacturonic acid and glucose in addition to small amounts of arabinose and galactose. This study demonstrated that CCP may enhance the innate immune responses and potentially improve the immune functions in the body.

Cinobufagin Suppresses Melanoma Cell Growth by Inhibiting LEF1.

Constitutive activation of the ß-catenin dependent canonical Wnt signaling pathway, which enhances tumor growth and progression in multiple types of cancer, is commonly observed in melanoma. LEF1 activates ß-catenin/TCF4 transcriptional activity, promoting tumor growth and progression. Although several reports have shown that LEF1 is highly expressed in melanoma, the functional role of LEF1 in melanoma growth is not fully understood. While A375, A2058, and G361 melanoma cells exhibit abnormally high LEF1 expression, lung cancer cells express lower LEF1 levels. A luciferase assay-based high throughput screening (HTS) with a natural compound library showed that cinobufagin suppressed ß-catenin/TCF4 transcriptional activity by inhibiting LEF1 expression. Cinobufagin decreases LEF1 expression in a dose-dependent manner and Wnt/ß-catenin target genes such as Axin-2, cyclin D1, and c-Myc in melanoma cell lines. Cinobufagin sensitively attenuates cell viability and induces apoptosis in LEF1 expressing melanoma cells compared to LEF1-low expressing lung cancer cells. In addition, ectopic LEF1 expression is sufficient to attenuate cinobufagin-induced apoptosis and cell growth retardation in melanoma cells. Thus, we suggest that cinobufagin is a potential anti-melanoma drug that suppresses tumor-promoting Wnt/ß-catenin signaling via LEF1 inhibition.

Ginsenoside metabolite 20(S)-protopanaxatriol from Panax ginseng attenuates inflammation-mediated NLRP3 inflammasome activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax ginseng C.A. Meyer (Araliaceae), has been used in traditional medicine for preventive and therapeutic purposes in Asian countries. One of the active ginsenoside metabolites, 20(S)-Protopanaxatriol (PPT), has been associated with diverse pharmacological effects, including anti-inflammatory properties. AIM OF THE STUDY: Although the capacity of PPT as an anti-inflammatory agent has been studied, this study aimed to explore the intrinsic mechanism of PPT in regulating inflammasome activation-mediated inflammatory responses in experimental models. MATERIALS AND METHODS: Lipopolysaccharide (LPS)-primed peritoneal macrophages in vitro was used to study the role of PPT on inflammasome activation. LPS-induced septic shock and monosodium urate (MSU)-induced murine peritonitis models were employed for in vivo evaluations. RESULTS: PPT attenuated NLRP3 inflammasome activation and also reduced ASC oligomerization, leading to attenuation of interleukin (IL)-1ß secretion. Further, PPT inhibited IL-1ß secretion in both LPS-induced septic shock and MSU-induced mouse peritonitis models. CONCLUSIONS: This study revealed that ginsenoside metabolite PPT, inhibits inflammation-mediated inflammasome activation and supported the traditional use of ginseng in treating various inflammatory disorders.

Molecular Targeting of VEGF with a Suramin Fragment-DOCA Conjugate by Mimicking the Action of Low Molecular Weight Heparins.

Molecular targeting of growth factors has shown great therapeutic potential in pharmaceutical research due to their roles in pathological conditions. In the present study, we developed a novel suramin fragment and deoxycholic acid conjugate (SFD) that exhibited the potential to bind to the heparin-binding site (HBD) of vascular endothelial growth factor (VEGF) and to inhibit its pathogenic action for the first time. Notably, SFD was optimally designed for binding to the HBD of VEGF using the naphthalenetrisulfonate group, allowing to observe its excellent binding efficacy in a surface plasmon resonance (SPR) study, showing remarkable binding affinity (KD = 3.8 nM) as a small molecule inhibitor. In the tubular formation assay, it was observed that SFD could bind to HBD and exhibit antiangiogenic efficacy by inhibiting VEGF, such as heparins. The cellular treatment of SFD resulted in VEGF-inhibitory effects in human umbilical vein endothelial cells (HUVECs). Therefore, we propose that SFD can be employed as a novel drug candidate to inhibit the pathophysiological action of VEGF in diseases. Consequently, SFD, which has a molecular structure optimized for binding to HBD, is put forward as a new chemical VEGF inhibitor.

The Molecular Links between Cell Death and Inflammasome.

Programmed cell death pathways and inflammasome activation pathways can be genetically and functionally separated. Inflammasomes are specialized protein complexes that process pro-inflammatory cytokines, interleukin-1ß (IL-1ß), and IL-18 to bioactive forms for protection from a wide range of pathogens, as well as environmental and host-derived danger molecules. Programmed cell death has been extensively studied, and its role in the development, homeostasis, and control of infection and danger is widely appreciated. Apoptosis and the recently recognized necroptosis are the best-characterized forms of programmed death, and the interplay between them through death receptor signaling is also being studied. Moreover, growing evidence suggests that many of the signaling molecules known to regulate programmed cell death can also modulate inflammasome activation in a cell-intrinsic manner. Therefore, in this review, we will discuss the current knowledge concerning the role of the signaling molecules originally associated with programmed cell death in the activation of inflammasome and IL-1ß processing.

Chrysanthemum indicum extract inhibits NLRP3 and AIM2 inflammasome activation via regulating ASC phosphorylation.

ETHNOPHARMACOLOGICAL RELEVANCE: Chrysanthemum indicum (C. indicum), a perennial plant, has long been used to treat inflammation-related disorders, such as pneumonia, hypertension, gastritis, and gastroenteritis. AIM OF THE STUDY: The inhibitory effect of C. indicum extract (C.I) on inflammasome activation was investigated to validate its potential in treating inflammation related disorders. MATERIALS AND METHODS: LPS-primed bone marrow-derived macrophages (BMDMs) were used to confirm the inhibitory effect of C.I on selective inflammasome activation in vitro. A monosodium urate (MSU)-induced murine peritonitis model was employed to study the effect of C.I in vivo. RESULTS: C.I inhibited activation of NLRP3 and AIM2 inflammasomes, leading to suppression of interleukin-1ß secretion in vitro. Further, C.I regulates the phosphorylation of apoptosis-associated speck-like protein containing a CARD (ASC), which could be the main contribution to attenuate these inflammasomes activation. C.I also suppressed secretion of pro-inflammatory cytokines and neutrophils recruitment in MSU-induced murine peritonitis model. CONCLUSIONS: This study provides scientific evidence substantiating the traditional use of C. indicum in the treatment of inflammatory diseases, including gout, which is induced by physiologically analogous cause to MSU-induced peritonitis.