Degradation Of Lipopolysaccharide Research Articles

Abstract 2461: Discovery of an orally available and liver-targeted ALPK1 agonist for cancer immunotherapy

Abstract Boosting anti-tumor immune responses can be achieved by activating the innate immune system. Despite the ongoing development of numerous STING and TLR agonists for this purpose, none of them have progressed to late-stage clinical evaluation. Alpha protein kinase-1 (ALPK1) is an intracellular innate immune receptor newly identified for its ability to detect ADP-heptose, a metabolite released during the biosynthesis and degradation of lipopolysaccharides (LPS). Since heptose is not naturally present in mammalian cells, the recognition of heptose metabolites by ALPK1 serves as a reliable mechanism to identify bacterial infections. When ADP-heptose binds to ALPK1, it initiates the phosphorylation of TIFA, thereby triggering a cascade of immune responses. Systemic administration with ADP-heptose induces robust ALPK1-dependent cytokine responses in mice. While high doses of ADP-heptose demonstrate anti-tumor effects, its instability and low in vivo potency hinder its clinical development. Through a SBDD medicinal chemistry effort, a novel, stable, and potent ADP-heptose analog, designated PTT-936, has been discovered and is progressing into clinical evaluation in cancer patients. PTT-936 exhibits remarkable nanomolar potencies for ALPK1 activation in various cellular assays. Notably, in human PBMCs, PTT-936 induces both concentration-dependent release of cytokines and activation of NK and T cells. Consistent with in vitro studies, oral administration of PTT-936 triggered significant cytokine responses in mice at microgram dose levels. However, overt toxicities were observed only at milligram dose levels, indicating a wide therapeutic window for this compound. In syngeneic tumor models, oral administration of PTT-936 induced dose-dependent tumor growth inhibition, which correlated with elevations in several cytokine biomarkers. Mechanistic studies show that PTT-936, working exclusively through the ALPK1 pathway, elicited strong innate and adaptive immune responses, as evidenced by robust cytokine induction and immune cell activation and recruitment. In syngeneic mouse models, the anti-tumor effects of PTT-936 were significantly hindered by either CD8+ T cell depletion or neutralization of select cytokines. Besides monotherapy studies, compelling evidence demonstrates strong synergistic anti-tumor effects when combining PTT-936 with immune checkpoint inhibitors. Intriguingly, PTT-936 exhibits favorable tolerability and a wider therapeutic index compared to certain clinical-stage STING and TLR agonists. Furthermore, PTT-936 does not enhance cytokine elevations induced by checkpoint inhibitors, which have been linked to adverse events (including CRS) observed in clinical combination studies. The dosing frequency of PTT-936 in the upcoming first-in-human clinical trial is carefully designed to prioritize both tolerability and patient compliance. Citation Format: Yupeng Wang, Nana Du, Xiaona Dong, Xuebing Sun, Yuan Hong, Xiaolong Yang, Zhijiang Ji, Qianyu Wang, Yun Lv, Zhichen Shi, Yajuan Gao, Xiaofu Hou, Yaxin Zhang, Juan Wang, Rui Mao, Yinxin Liu, Liang Li, Jingjin Ding, Hexiang Wang, Nan Hu, Counde O’Yang, Zhihong Li, Keith Bley, Feng Shao. Discovery of an orally available and liver-targeted ALPK1 agonist for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2461.

Wheat phytase can alleviate the cellular toxic and inflammatory effects of lipopolysaccharide.

The objective of this study was to characterize the enzymatic hydrolysis of lipopolysaccharide (LPS) by wheat phytase and to investigate the effects of wheat phytase-treated LPS on in vitro toxicity, cell viability and release of a pro-inflammatory cytokine, interleukin (IL)-8 by target cells compared with the intact LPS. The phosphatase activity of wheat phytase towards LPS was investigated in the presence or absence of inhibitors such as L-phenylalanine and L-homoarginine. In vitro toxicity of LPS hydrolyzed with wheat phytase in comparison to intact LPS was assessed. Cell viability in human aortic endothelial (HAE) cells exposed to LPS treated with wheat phytase in comparison to intact LPS was measured. The release of IL-8 in human intestinal epithelial cell line, HT-29 cells applied to LPS treated with wheat phytase in comparison to intact LPS was assayed. Wheat phytase hydrolyzed LPS, resulting in a significant release of inorganic phosphate for 1 h (p < 0.05). Furthermore, the degradation of LPS by wheat phytase was nearly unaffected by the addition of L-phenylalanine, the inhibitor of tissue-specific alkaline phosphatase or L-homoarginine, the inhibitor of tissue-non-specific alkaline phosphatase. Wheat phytase effectively reduced the in vitro toxicity of LPS, resulting in a retention of 63% and 54% of its initial toxicity after 1–3 h of the enzyme reaction, respectively (p < 0.05). Intact LPS decreased the cell viability of HAE cells. However, LPS dephosphorylated by wheat phytase counteracted the inhibitory effect on cell viability. LPS treated with wheat phytase decreased IL-8 secretion from intestinal epithelial cell line, HT-29 cell to 14% (p < 0.05) when compared with intact LPS. In conclusion, wheat phytase is a potential therapeutic candidate and prophylactic agent for control of infections induced by pathogenic Gram-negative bacteria and associated LPS-mediated inflammatory diseases in animal husbandry.

Insights into electrochemical decomposition mechanism of lipopolysaccharide using TiO2 nanotubes arrays electrode

Electrochemical decomposition of lipopolysaccharide (LPS) was firstly investigated over titania nanotubes (TNTs) arrays electrode. The TNTs layer of this electrode consisted of numerous tubular structures which arranged tightly, and the average diameter of each nanotube is 100 ± 5 nm. The degradation of LPS and polysaccharides followed pseudo-first-order kinetics. The optimal LPS removal ratio was nearly 80 %. The endotoxin toxicity of LPS steadily decreased during the electrolysis process. The acute toxicity of the intermediates increased suddenly at the beginning of electrochemical degradation process (< 5 min), then maintained high inhibition ratio (> 95 %) for about 150 min, and decreased significantly (< 10 %) after electrolysis for 240 min. After 20 min of electrolysis, LPS with molecular weight of 116,854 Da was transformed into small molecular compounds with molecular weights of 59,312 - 12,209 Da. Possible degradation and detoxification mechanisms of LPS including electric-field-force-driving accumulation, adsorption and direct electron transfer on TNTs arrays electrode, and •OH oxidation were proposed. This study underscores that electrochemical technique can be applied to eliminate and decrease the toxicity of LPS from contaminated water.

A new roller conveyer system of non-thermal gas plasma as a potential control measure of plant pathogenic bacteria in primary food production

We have developed a novel roller conveyer plasma device, which generates gas plasma via atmospheric pressure dielectric barrier discharge (APDBP) on rollers. High voltage and earth electrodes are incorporated into the rollers, which are covered with a silicon sheet. Bactericidal activity of the device was measured against Xanthomonas campestris pv. campestris (Xcc), which was spotted onto aluminium plates and subjected to gas plasma treatment generated by APDBD. After treatment, viable cell number of Xcc decreased with a decimal reduction time (D value) of 0.90 min (initial population: 5.0 × 108 CFU/ml) and below 0.34 min (initial population: 9.8 × 105 CFU/ml). APDBD treatment induced slight morphological changes to Xcc along with significant degradation of lipopolysaccharides (LPS) and degradation/oxidation of genomic DNA to form 8-hydroxy-2'-deoxyguanosine (8-OHdG). Reactive chemical products (hydrogen peroxide, nitrite and nitrate), ultraviolet light (UV-A) and slight temperature elevations were observed during operation of the device. Our results suggest hydrogen peroxide and nitrite generated during operation of the device cause oxidation and degradation of the genomic DNA as well as the degradation of LPS, which are potential mechanisms for inactivation. In addition, the successful inactivation of Xcc on cabbage leaf by the device suggests the method could have practical applications.

Structure of the O-polysaccharide of Providencia alcalifaciens O2 containing ascarylose and N-(l-alanyl)-d-glucosamine

The O-polysaccharide was obtained by degradation of the lipopolysaccharide of Providencia alcalifaciens O2 under mild acidic conditions followed by GPC. The polysaccharide was found to contain two unusual components: 3,6-dideoxy-l-arabino-hexose (ascarylose, Asc) and 2-(l-alanyl)amino-2-deoxy-d-glucose (GlcNAla). Ascarylose was partially split off during lipopolysaccharide degradation and could be eliminated completely by selective acid hydrolysis, which also partially cleaved the β-GalNAc-(1→6) linkage. The following structure of the branched pentasaccharide repeating unit was established by 1H and 13C NMR spectroscopy of the O-polysaccharide and O-deacetylated polysaccharide, as well as products of partial acid hydrolysis: [Display omitted]

Bacterial antigen polysaccharides. 43. Structure for the O-specific polysaccharide of Providencia alcalifaciens O46

Acid O-specific polysaccharide containing D-glucose, D-glucuronic acid, L-fucose, and 2-acetamido-2-deoxy-D-glucose was obtained by mild acid degradation of lipopolysaccharide from Providencia alcalifaciens O46. Consideration of the data revealed the following structure of the hexasaccharide repeating unit of O-specific polysaccharide under methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H, (1)H-COSY, TOCSY-, ROESY-,(1)H, (13)C-HSQC-, and HMQC-TOCSY experiments: [Formula: see text].

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It has been supposed that lipopolysaccharide (LPS) derived from the gram negative bacteria of subgingival plaque is one of the important etiologic factors in periodontal disease. The purpose of this study was to detect specifically the LPS from Bacteroides gingivalis (Bg) and to determine the effects of gram-positive bacteria on LPS in culture supernatant of Bg. Enzyme-linked immunosorbent assay was used for specific detection of LPS from Bg. LPS of Bg could be measured in concentrations as low as 4 micrograms/ml. LPS of Bg was not detected in gingival crevicular fluid from periodontal disease patients. There were no significant differences in the concentration of LPS between the culture supernatant of Bg and co-cultivation of Bg and gram-positive bacteria. In this study, gram-positive bacteria had no effects on release and degradation of LPS in the culture supernatant of Bg.

The fate of E. coli lipopolysaccharide after the uptake of E. coli by murine macrophages in vitro.

The fate of bacterial lipopolysaccharide (LPS) after the uptake of Escherichia coli by macrophages in vitro was studied. The LPS of the galactose epimerase-deficient E. coli J5 mutant was specifically radiolabeled with [3H]galactose by growing the organism in a basic salts medium containing galactose. Control bacteria were uniformly radiolabeled by growth in [14C]glucose and unlabeled galactose-containing medium. Surface constituents of E. coli were also labeled with 125I. After in vitro phagocytosis of labeled E. coli by murine peritoneal exudate macrophages, the rate of exocytosis of LPS, as assessed by release of 3H over a 72-hr period, was considerably reduced in comparison with other bacterial constituents (14C and 125I release). The [3H]galactose-labeled material exocytosed from macrophages and that remaining intracellularly (obtained from macrophage lysates) were isolated by cesium chloride (CsCl) density gradients and were shown to have altered density profiles as compared with purified E. coli LPS. The macrophage-"processed" [3H] galactose-containing fractions from CsCl density gradients of culture supernatants or macrophage lysates were capable of clotting Limulus amebocyte lysate. The [3H]galactose material obtained from 48-hr macrophage lysates and culture supernatants could also induce a lethal response in actinomycin D-treated mice. These data suggest that bacterial LPS may be selectively retained by the macrophage and that the post-phagocytic events that result in bacterial degradation are not accompanied by the degradation of LPS. Furthermore, although the LPS may be modified by the macrophage, it retains its biologic activity.