HMGB1 (high mobility group protein) has been established as a key inflammatory mediator associated with various chronic diseases, particularly in neuroinflammatory pathogenesis. In this work, the potential of chrysin (Chr)/ apigenin (Api)/luteolin (Lut) as natural functional food ingredients was evaluated on their interactions with HMGB1 through integrated multi-spectroscopic analysis. Surface plasmon resonance (SPR) results of Api and Lut showed the dissociation constants (KD) values of 2.991 x 10-5 M and 3.206 x 10-6 M, respectively, which indicated that the hydroxyl group on the B-ring of flavonoids may impact their binding affinity with HMGB1. Fluorescence spectroscopy and circular dichroism (CD) analyses revealed that the binding of three flavonoids to HMGB1 caused static quenching and significantly altered their spatial conformation, resulting in a reduction in the alpha-helix content from 60.71 f 1.30% to Chr(44.24 f 1.27%), Api(47.52 f 2.79%) and Lut(49.20 f 2.07%), respectively. The synchronous and three-dimensional fluorescence spectrum further revealed that these interactions led to the microenvironmental changes in the chromogenic amino acids in HMGB1. Molecular docking experiments indicated that hydrogen bonding was the primary force between the interaction of three flavonoids and HMGB1, and the key amino acids forming the hydrogen bonds included Lys94, Lys95, Asp98, and Arg104 residues. Furthermore, the three flavonoids could mitigate HMGB1-induced neuroinflammation on BV2 microglia cells in the bioassay. Due to the key role of HMGB1 in neuroinflammation and neurological disorders, Chr, Api, and Lut could be applied to improve the treatment of neurological diseases, and they also provided new insights for the development of new dietary supplements of flavonoids.

High mobility group box 1 (HMGB1) is a nonhistone chromatin protein predominantly located in the nucleus. However, under pathological conditions, HMGB1 can translocate from the nucleus to the cytoplasm and subsequently be released into the extracellular space through both active secretion and passive release mechanisms. The distinct cellular locations of HMGB1 facilitate its interaction with various endogenous and exogenous factors, allowing it to perform diverse functions across a range of diseases. This Perspective provides a comprehensive overview of the structure, release mechanisms, and multifaceted roles of HMGB1 in disease contexts. Furthermore, it introduces the development of both small molecule and macromolecule inhibitors targeting HMGB1 and its interaction with receptors. A detailed analysis of the predicted pockets is also presented, aiming to establish a foundation for the future design and development of HMGB1 inhibitors.

Introduction HMGB1 is a non-histone chromatin protein released or secreted from cells in response to tissue damage or infection. Extracellular HMGB1, as a crucial immunomodulatory factor, binds with several different receptors to innate inflammatory responses that aggravate acute and chronic liver diseases. The increased levels of HMGB1 have been reported in various liver diseases, highlighting that it represents a potential biomarker and druggable target for therapeutic development.

IntroductionHMGB1 is a non-histone chromatin protein released or secreted in response to tissue damage or infection. Extracellular HMGB1, as a crucial immunomodulatory factor, binds with several different receptors to innate inflammatory responses that aggravate acute and chronic liver diseases. The increased levels of HMGB1 have been reported in various liver diseases, highlighting that it represents a potential biomarker and druggable target for therapeutic development.Areas coveredThis review summarizes the current knowledge on the structure, function, and interacting receptors of HMGB1 and its significance in multiple liver diseases. The latest patented and preclinical studies of HMGB1 inhibitors (antibodies, peptides, and small molecules) for liver diseases are summarized by using the keywords 'HMGB1,' 'HMGB1 antagonist, HMGB1-inhibitor,' 'liver disease' in Web of Science, Google Scholar, Google Patents, and PubMed databases in the year from 2017 to 2023.Expert opinionsIn recent years, extensive research on HMGB1-dependent inflammatory signaling has discovered potent inhibitors of HMGB1 to reduce the severity of liver injury. Despite significant progress in the development of HMGB1 antagonists, few of them are approved for clinical treatment of liver-related diseases. Developing safe and effective specific inhibitors for different HMGB1 isoforms and their interaction with receptors is the focus of future research.

Using the unique triterpenoid probe, we found that the binding interface of HMGB1 was responsible for the recognition of TLR4 located on the “L” shaped B-box with K114 as a crucial hot-spot residue.

In the current study, tea saponin, identified as the primary bioactive constituent in seed pomace of Camellia oleifera Abel., was meticulously extracted and hydrolyzed to yield five known sapogenins: 16-O-tiglogycamelliagnin B (a), camelliagnin A (b), 16-O-angeloybarringtogenol C (c), theasapogenol E (d), theasapogenol F (e). Subsequent biotransformation of compound a facilitated the isolation of six novel metabolites (a1-a6). The anti-inflammatory potential of these compounds was assessed using pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns molecules (DAMPs)-mediated cellular inflammation models. Notably, compounds b and a2 demonstrated significant inhibitory effects on both lipopolysaccharide (LPS) and high-mobility group box 1 (HMGB1)-induced inflammation, surpassing the efficacy of the standard anti-inflammatory agent, carbenoxolone. Conversely, compounds d, a3, and a6 selectivity targeted endogenous HMGB1-induced inflammation, showcasing a pronounced specificity. These results underscore the therapeutic promise of C. oleifera seed pomace-derived compounds as potent agents for the management of inflammatory diseases triggered by infections and tissue damage.

Functional regulation of proteins by ligand-protein interactions plays a crucial role in understanding biological processes and identifying potential drugs. High mobility group box 1 (HMGB1) plays a pivotal role in sterile inflammation as a key immunomodulatory protein. Genistein, a well-known isoflavone compound, has been shown to have neuroprotective effects. In this study, we investigated the genistein-HMGB1 interactions using experimental and computational approaches. Our results revealed that genistein binds to HMGB1 with a KD value of 6.06 × 10-5 M. The addition of genistein significantly quenched the fluorescence of HMGB1. Thermodynamic analyses demonstrated that hydrogen bonds and hydrophobic forces are the primary forces during the binding process. Furthermore, the interaction between genistein and HMGB1 led to changes in the microenvironment of protein chromogenic amino acids and subtle alterations in the protein secondary structure. Molecular modeling results indicate that Pro95, Pro98, and Lys154 are the major amino acid residues for genistein binding to HMGB1. Meanwhile, at the cellular level, an inhibitory effect of genistein on HMGB1-induced NO release from microglia was observed, demonstrating an inhibition rate of 42.1 %. Our studies demonstrated that genistein could be applied in treating neurological diseases through its interaction with HMGB1.

The microbial transformation of maslinic acid (MA) was investigated for the screening of potential metabolites as anti-inflammatory food supplements. Six novel and two known metabolites were obtained. In the bioassay of lipopolysaccharide and high mobility group box-1 protein (HMGB1) stimulated nitric oxide production in RAW 264.7 cells, it was surprising to see that most of the metabolites showed selective inhibitory effects on the HMGB1 model. Among them, metabolites 6 (2a,3fi,7fi,21fi- tetrahydroxy-olean-12-en-28-oic acid) and 8 (2a,3fi,23-trihydroxy-olean-12-en-28,29-dioic acid) exhibited the IC50 values of 10.77 +/- 2.87 and 11.52 +/- 1.09 mu M, respectively. Molecular docking further revealed the newly introduced hydroxyl groups on C-21/C-23 and a carboxyl group on C-29 via biotransformation may interact with the amino residue of Ser12, Lys39, and Glu43 in HMGB1. Thus, biotransformation of MA or related extracts would provide an applicable routine for obtaining dietary supplements that selectively target damage-associated molecular patterns that stimulate sterile inflammation.

Background High mobility group box 1 protein (HMGB1), a lethal late inflammatory mediator, contributes to the pathogenesis of diverse inflammatory and infectious diseases. Astragaloside IV and calycosin as active ingredients in Astragalus membranaceus , possess potent regulatory ability on HMGB1-induced inflammation, however, the interaction between these two phytochemicals and HMGB1 has not been elucidated yet. Methods To further investigate the interaction of astragaloside IV, calycosin with HMGB1 protein, surface plasma resonance (SPR) and a series of spectroscopic methods, including UV spectra, fluorescence spectroscopy, circular dichroism (CD), were used. Molecular docking was also carried out to predict the atomic level’s binding modes between two components and HMGB1. Results Astragaloside IV and calycosin were found to be able to bind HMGB1 directly and affect the secondary structure and environment of the chromogenic amino acids of HMGB1 to different extents. In silico , astragaloside IV and calycosin showed a synergistic effect by binding to the two independent domains B-box and A-box in HMGB1, respectively, where hydrogen and hydrophobicity bonds were regarded as the crucial forces. Conclusion These findings showed that the interaction of astragaloside IV and calycosin with HMGB1 impaired its proinflammatory cytokines function, providing a new perspective for understanding the mechanism of A. membranaceus in treating aseptic and infectious diseases.

Ursolic acid (UA) is a pentacyclic triterpenoid widely found in in medicinal plants, edible plants, fruits, and flowers. The great interest in this bioactive compound is related to the positive effects in human health. However, its limited solubility, moderate biological activity and poor bioavailability limit the potential and further applications of UA. Here, we explored the efficacy of MeON-Glycosides of UA in inhibiting tumor cell proliferation. A number of compounds showed significant antitumor activity against tested five cancer cell lines. Among them, compound 2a exhibited the most potent activity against HepG2 cells with IC50 values of 3.1 ± 0.5 μM. Especially, compound 2a could induce HepG2 cells apoptosis and reduce mitochondrial membrane potential. Western blot analysis showed that compound 2a up-regulated Bax, cleaved caspase-3/9, cleaved PARP levels and down-regulated Bcl-2 level of HepG2 cells. These results indicated that compound 2a could obviously induce the apoptosis of HepG2 cells. At the same time, compound 2a significantly decreased the expression of p-AKT and p-mTOR, which indicated that compound 2a might exert its cytotoxic effect by targeting PI3K/AKT/mTOR signaling pathway. Moreover, the in silico ADME predictions showed that compound 2a has improved water solubility and other properties. Thus, compound 2a may be a promising antitumor candidate, which may be potentially used to prevent or treat cancers.

Pentacyclic triterpenoids are considered to be the potential HMGB1 inhibitors, but due to the limited number of hydrogen bond donors and the number of rotatable bonds in the rigid skeletons, their further chemical biology research with this target was restricted. To improve these profiles, microbial-catalyzed Baeyer-Villiger oxidation of the primary ursane and oleanane-type triterpenoids including uvaol (1), erythrodiol (2), oleanolic acid (3), and ursolic acid (4) was performed by Streptomyces olivaceus CICC 23628. As a result, ten new and one known A-ring cleaved metabolites were obtained and the possible biogenetic pathways were also discussed based on the HPLC-MS analysis. Furthermore, the direct interactions between compounds 1d, 2b, and HMGB1 were observed by the biolayer interferometry technique. Molecular docking revealed that the newly introduced vicinal diol at C-4, C-24, and the hydroxyl group at C-21 of compound 1d are crucial for binding with HMGB1. The cellular assay showed that co-treatment of 1d could significantly block HMGB1-activated nitric oxide release with an IC50 value of 9.37 μM on RAW 264.7 cells. Altogether, our research provides some insights into 3,4-seco-triterpenes as potential anti-inflammatory candidates for the discovery of novel HMGB1 inhibitors.

The intake of quercetin and its glycosides has been proven to effectively reduce the level of high mobility group protein 1 (HMGB1) and the degree of inflammation, but the underlying structural mechanism is still unclear. In this study, the direct interaction between HMGB 1 and quercetin, isoquercitrin, and rutin was recorded by surface plasmon resonance (SPR). The binding interactions led to the intrinsic fluorescence quenching of HMGB1 through static quenching mechanism based on fluorescence spectroscopy. Circular dichroism (CD) spectra showed a decrease in a-helical content of HMGB1 with a slight impact on the thermal stability of HMGB1. Furthermore, molecular simulations displayed three flavonoids-HMGB1 complexes maintained primarily by hydrogen bond and hydrophobic force, confirming the strong association with Phe14. Besides, co-treatment of quercetin could significantly ameliorate HMGB1-stimulated nitric oxide release in RAW264.7 cells. These intrinsic characteristics on the interaction of quercetin, isoquercitrin, and rutin with HMGB1 protein could be conducive to understanding the molecular mechanisms of flavonoids in HMGB1-related inflammatory diseases.

In the biosynthetic pathway of pentacyclic triterpenes (PTs), tailoring reactions can produce a wide range of end products from a small number of common scaffolds and the microbial transformation has also been established as an alternative technique for this purpose. In this study, we explored the tailoring reactions involved in the microbial transformation of pentacyclic triterpenes by Penicillium griseofulvum CICC 40293. Preparative biotransformation of eight different PTs from three scaffolds resulted in the isolation of thirteen metabolites. The structures of metabolites were elucidated by HR-ESI-MS, 1D, and 2D NMR spectroscopy. We discovered the highly efficient regio- and stereo-selective hydroxylation of inactivated sp(3) CH2 and CH3 on the position of 2 alpha, 7 beta, 15 alpha, 2 beta, 23(angular methyl), and 30-COOH glycosylation, this versatile tailoring system for PTs would provide an effective method for expanding their structural diversities. In addition, all compounds were subjected to the bioassay on the model of lipopolysaccharide (LPS)-stimulated RAW 264.7 cells to evaluate their anti-inflammatory activity through nitric oxide (NO) inhibition activity. Compounds 2a and 5a exhibited excellent NO inhibitory activity with IC50 values of 8.35 +/- 2.81 mu M and 19.60 +/- 4.25 mu M, respectively.

For the discovery of new pentacyclic triterpenes as a potential anti-inflammatory agent, microbial transformation of uvaol by Penicilium griseofulvum CICC 40293 and Streptomyces griseus ATCC 13273 was investigated. Stereoselective hydroxylation and epoxidation reactions were observed in the biotransformation. Moreover, six new metabolites were isolated and structurally elucidated by HR-ESI-MS and NMR spectrum. All the compounds were evaluated upon the inhibitory effects of nitric oxide (NO) release in RAW 264.7 cells induced by lipopolysaccharide (LPS) and high-mobility group box 1 (HMGB1). Among them, compound 3 (13, 28-epoxy-3β, 7β, 21β-trihydroxy-urs-11-ene) with the unique epoxy structure and compound 5 (3β, 21β, 24, 28-tetrahydroxy-urs-12-en-30-oic acid), exhibited a considerable inhibitory effect on both models while compound 2 (urs-12-ene-3β, 7β, 21β, 28-tetraol) showed a significant bias in the LPS-induced inflammatory response with IC50 value of 2.22 μM. Therefore, this study could provide some insights on the discovery of the pentacyclic triterpene leads for the treatment of either DAMPs or PAMPs triggered inflammation.

For the optimum use of soyasaponins isolated from soybean cake and to explore the potential anti-inflammatory agents from pentacyclic triterpenes as natural food supplements, microbial transformation of soyasapogenol A was carried out. Four strains of microbes, including Bacillus megaterium CGMCC 1.1741, Penicillium griseofulvum CICC 40293, Bacillus subtilis ATCC 6633, and Streptomyces griseus ATCC 13273, showed robust catalytic capacity to the substrate. Preparative biotransformation and column chromatographic purification led to the isolation of 10 novel and 1 reported metabolites. The structure elucidation was performed using 1D/2D NMR and HR-ESI-MS analytical method. Several novel tailoring reactions, such as allyl oxidation, C-C double bond rearrangement, hydroxylation, dehydrogenation, and glycosylation, were observed in the biotransformation. In the follow-up bioassay, most of the metabolites exhibited low cytotoxicity and potent inhibitory activity against the production of nitric oxide (NO) in RAW 264.7 cells stimulated by lipopolysaccharide. Especially compound 6 (3-oxo-11α,21β,22β,24-tetrahydroxy-olean-12-ene) showed comparable activity to the positive control of quercetin with an IC50 value of 16.70 μM. These findings provided an experimental approach to achieve the derivatization of natural aglycons in soybeans through microbial transformation for developing potent anti-inflammatory food supplements.

High mobility group box-1 protein (HMGB1) is a typical Damage-Associated Molecular Patterns (DAMPs) released in response to cellular inflammation. The pentacyclic triterpenes (PTs) are considered to be the natural inhibitors against HMGB1-related inflammation. To explore new lead compounds of PTs as anti-inflammatory agents, biotransformation of four PTs by Streptomyces olivaceus CICC 23628 was investigated in this study. As a result, thirteen unique 3,4-seco-triterpenes metabolites were isolated and twelve of them were first identified and reported. Structures of metabolites were determined based on HR-ESI-MS, 1D/2D NMR, and single-crystal X-ray diffraction. Furthermore, all compounds were subjected to the bioassay on the model of HMGB1-stimulated RAW 264.7 cells to evaluate their anti-inflammatory activity through nitric oxide (NO) inhibition activity. Compounds 3b (3,4-seco-olean-12-en-4,21 alpha,22 beta,24-tetrahydroxy-ol-3-oic acid) and 2b (3,4-seco-olean-12-en4,21 beta,22 beta,24,29-pentahydroxy-ol-3-oic acid) exhibited NO inhibitory activity with IC50 values of 15.94 mu M and 36.00 mu M, respectively. Thus, indicating their potential as HMGB1 inhibitors and in developing potent anti-inflammatory agents. This work provides an operationally simple, efficient method for the rapid diversification of the PTs scaffold for a variety of distinctive 3,4-seco-triterpenes to facilitate the discovery of potential anti-inflammatory compounds.

In this study, we described the microbial catalyzed allylic oxidation by Bacillus megaterium CGMCC 1.1741 of three Δ12-pentacyclic triterpenes, erythrodiol (1), uvaol (2), hederagenin (3) and of four steroids including Δ5-steroids, diosgenin (4), pennogenin (5), 25(R,S)-ruscogenin (6) and Δ4-steroid, diosgenone (7). As a result, fourteen metabolites were generated with allyl hydroxyl moiety. Ten (1a-c, 2a, 2c, 3a, 5a-b, and 6a-b) of them were new natural products and their structures were determined on the basis of 1D/2D NMR and HR-MS data. Biocatalytic allylic oxidation by B. megaterium CGMCC 1.1741 is thus a potential non-toxic and efficient alternative method toward metal-mediated oxidation procedures in the synthesis of natural products and medicines.

Glycyrrhetinic acid (GA), the major bioactive pentacyclic triterpene aglycone of licorice root, was known to play a vital role in anti-ulcer, anti-depressant, anti-inflammatory, and anti-allergic. In this study, we semi-synthesized five GA derivatives by a series of chemical reactions. They were selected as substrates for the biotransformation and yielded thirteen metabolites by Bacillus subtilis ATCC 6633 and Bacillus megaterium CGMCC 1.1741. Their structures were identified on the basis of extensive spectroscopic methods and nine of them were found for the first time. Two main types of reactions, regio- and stereo-selective hydroxylation and glycosylation, especially in the unactivated C-H bonds including C-11, C-19 and C-27, were observed in the biotransformation process, which greatly expand the chemical diversities of GA derivatives. All compounds were tested for their inhibitory effects on nitric oxide (NO) generation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Among them, olean-12-ene-3β,7β,15α,19α,30-pentol (16) and olean-12-ene-3β,7β,15α,27,30-pentol (17) showed significant inhibitory effect with IC50 values of 0.64 and 0.07 μM, respectively.

Erythrodiol, a typical pentacyclic triterpenic diol in olive oil and its byproduct, olive pomace, frequently appears in food additives for the prevention of cardiovascular diseases because of its antioxidation, anti-inflammatory, and antitumor activities. To develop new derivatives of erythrodiol (1), preparative biotransformations were investigated through Streptomyces griseus ATCC 13273, Penicilium griseofulvum CICC 40293, and Bacillus subtilis ATCC 6633, and ten new (1a-1j) and one known metabolites were isolated. Their structures were elucidated by high resolution electrospray ionization mass spectrometry (HR-ESI-MS) and one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy. Furthermore, relative to 1, most metabolites exhibited lower toxicity and more potent inhibitory activities against nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. In particular, the glycosylated metabolite 1k showed a dramatically increased inhibitory effect with an IC50 value of 2.40 mu M, which is even lower than that of quercetin. Thus, biotransformation of erythrodiol is a viable strategy for discovering new triterpenes as food supplements with anti-inflammatory properties.