Biology subjects

Schachter, J.

Publications and source records attributed to Schachter, J..

5 recordsLinked to original sources

bioRxiv2025

ND-13, a DJ-1 derived peptide, as a novel pharmacological approach in the prevention of NLRP3 inflammasome activity in Diabetic Nephropathy

Diabetic nephropathy is the most important cause of renal failure worldwide and is characterized by sustained inflammation regulated in part by NLRP3 Inflammasome. Attenuation of inflammation is a major priority to prevent renal damage. Our previous publications show that DJ-1 has antioxidant and anti-inflammatory properties in the kidney. ND-13 is a short peptide consisting of 13 amino acids of the DJ-1-protein, which could increase DJ-1 pathway activation. The aim of these studies was to determine the role of NLRP3 in the pathogenesis of diabetic nephropathy and to study the possible renal protective effects of the DJ-1 pathway in diabetic mice and on inflammasome regulation. Bone marrow derived macrophages were treated with ND-13 and cultivated in high and low glucose. Diabetes was induced in C57Bl/6 mice via injection of streptozotocin and treated with ND-13 and MCC950, an inhibitor of the NLRP3 inflammasome. Peripheral mononuclear cells were isolated from human with diabetes, diabetic nephropathy and healthy donors and were plated, pretreated with ND-13 and stimulated with LPS+ATP. IL-1{beta} concentration in the medium of bone marrow derived macrophages increased by NLRP3 inflammasome stimulation by LPS+ATP, and decreased in macrophages pre-treated with ND-13, however, in the presence of LPS+Nigericin no effect was found. Peritoneal macrophages from diabetic mice were obtained and plated. Streptozotocin-induced diabetic C57BL/6 mice have increased the peritoneal cells IL-1{beta} production compared with control mice, suggesting that inflammasome may be activated in macrophages during diabetes and that ND-13 treatment normalized its activity. ND-13 and MCC950 decreased histologic evidence of tubular injury in STZ-induced diabetes in mice, additionally, significantly increased the mRNA expression of Col-I, Col-II, Tgf-{beta}, Il-6, Tnf- and P2x7 in the renal cortex, which was partially prevented by ND-13 and MCC950 pre-treatment. P2x7 mRNA expression was also increased in peritoneal macrophages obtained from diabetic mice, and its expression was attenuated by ND-13 pre-treatment. Peripheral mononuclear cells isolated from patients blood were plated and stimulated with LPS+ATP. Patients with diabetic nephropathy presented a significant increase of IL-1{beta} release compared to diabetic individuals and ND-13 could has a role in the prevention of inflammasome activation in healthy patients. Our results demonstrate that activation of the DJ-1 pathway is a promising approach to prevent renal inflammation and fibrosis during diabetes, by ameliorating inflammasome activation in peripheral immune cells. Thus, ND-13 could be a promising new therapeutic approach to attenuating inflammation and renal damage in diabetic nephropathy.

cell biology↗

bioRxiv2024

Gasdermin D mediates a fast transient release of ATP after NLRP3 inflammasome activation before ninjurin 1-induced lytic cell death

Pyroptosis is a lytic cell death triggered by the cleavage of gasdermin (GSDM) proteins and subsequent pore formation by the N-terminal domain oligomerization in the plasma membrane. GSDMD is cleaved by caspase-1/-4/-5/-11 upon inflammasome activation and mediates IL-1{beta} and IL-18 release. GSDMD pores favors ninjurin-1 (NINJ1) induced plasma membrane rupture and cell death. Here, we demonstrate that GSDMD mediates early ATP release upon NLRP3 inflammasome activation independently of NINJ1, occurring before IL-1{beta} release and cell death and constituting an early danger signal. The release of ATP is a transient signal terminated before the cells continue to permeabilize and die. The different N-terminal of GSDMA to E are also able to release ATP and induce monocyte migration towards pyroptotic cells. This study reveals ATP release as an early, and transient danger signal depending on GSDMD plasma membrane permeabilization, independently of the late stages of lytic cell death.

immunology↗

bioRxiv2024

Novel immunotherapy for multiple solid cancers using an Anti-HVEM blocking monoclonal antibody

INTRODUCTIONImmune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, yet their efficacy remains limited. Therefore, there is a clear need for new anti-tumor agents. HVEM (Herpes Virus Entry Mediator) plays a regulatory role in immunity, making it a promising cancer therapeutic target. EXPERIMENTAL DESIGNWe have developed Anti-4CB1, a fully human HVEM-BTLA and HVEM-CD160 blocking mAb and tested its anti-tumor activity in various in-vitro, ex-vivo and in-vivo models, alone or in combination with Anti-PD1. Finally, we analyzed HVEM expression in serum samples and melanoma tumor tissues and its correlation with HVEM and PD1 blockade treatment response. RESULTSIn-vitro assays demonstrated enhanced melanoma cell killing by autologous TILs in the presence of Anti-4CB1. In addition, Anti-4CB1 significantly increased cytotoxicity by up to 233% in a variety of ex-vivo cancer tissue samples of different indications. Notably, Anti-4CB1 demonstrated effectiveness in samples where Anti-PD1 was ineffective. In addition, significant anti-tumor activity at 10 mg/kg in combination with Anti-PD1 (TGI 95% p=0.0001) or at 25 mg/kg as monotherapy (TGI 50% p=0.0096) was demonstrated in a colon carcinoma transgenic mice model. Moreover, significant anti-tumor activity was observed in a mouse ImmunoGraft model (TGI 53% p=0.0102 as monotherapy). Finally, we demonstrated that HVEM expression correlated with response to Anti-HVEM and Anti-PD1 treatments. DISCUSSIONWe describe the development of Anti-4CB1, a fully human anti-HVEM mAb, blocking HVEM-BTLA and HVEM-CD160 human interactions and enhancing lymphocytes cytotoxicity against various cancer cells ex-vivo. In-vivo, Anti-4CB1 showed promising anti-tumor effects, particularly in combination with Anti-PD1, suggesting its therapeutic potential. Finally, HVEM expression may serve as a predictive marker for Anti-HVEM and Anti-PD1 treatments response, offering potential diagnostic utility in patient selection for these immunotherapies. These results support Anti-4CB1 potential as an anti-cancer therapeutic agent. Translational RelevanceThe discovery of immune checkpoint inhibitors (ICIs), marks a significant breakthrough in cancer therapy. However, despite the remarkable success of current ICIs targeting CTLA4 and PD1/PD-L1 pathways, a significant proportion of patients fail to respond or develop resistance. Therefore, the search for novel ICIs is crucial for advancing cancer immunotherapy. Anti-HVEM emerged as a promising candidate, as we demonstrate in ex-vivo and in-vivo models, its ability to block the HVEM-BTLA interaction, resulting in enhanced antitumor immune responses in solid tumors, alone or in combination with existing ICIs, to improve treatment outcomes. Moreover, the correlation between HVEM expression and treatment responses highlight its potential as an attractive target for biomarker-driven therapies. Investigating HVEM as an ICI offers a promising path to expand therapeutic options, personalize treatment approaches, address the evolving challenges in cancer immunotherapy and improve patient outcomes.

cancer biology↗

bioRxiv2024

Tryptanthrin Analogs Substoichiometrically Inhibit Seeded and Unseeded Tau4RD Aggregation

Microtubule-associated protein tau is an intrinsically disordered protein (IDP) that forms characteristic fibrillar aggregates in several diseases, the most well-known of which is Alzheimers disease (AD). Despite keen interest in disrupting or inhibiting tau aggregation to treat AD and related dementias, there are currently no FDA-approved tau-targeting drugs. This is due, in part, to the fact that tau and other IDPs do not exhibit a single well-defined conformation but instead populate a fluctuating conformational ensemble that precludes finding a stable "druggable" pocket. Despite this challenge, we previously reported the discovery of two novel families of tau ligands, including a class of aggregation inhibitors, identified through a protocol that combines molecular dynamics, structural analysis, and machine learning. Here we extend our exploration of tau druggability with the identification of tryptanthrin and its analogs as potent, substoichiometric aggregation inhibitors, with the best compounds showing potencies in the low nanomolar range even at a [~]100-fold molar excess of tau4RD. Moreover, conservative changes in small molecule structure can have large impacts on inhibitory potency, demonstrating that similar structure-activity relationship (SAR) principles as used for traditional drug development also apply to tau and potentially to other IDPs.

biochemistry↗

bioRxiv2023

Dynamic recycling of extracellular ATP in human epithelial intestinal cells

Intestinal epithelial cells play important roles in the absorption of nutrients, secretion of electrolytes and food digestion. The function of these cells is strongly influenced by purinergic signalling activated by extracellular ATP (eATP) and other nucleotides. The activity of several ecto-enzymes determines the dynamic regulation of eATP. In pathological contexts, eATP may act as a danger signal controlling a variety of purinergic responses aimed at defending the organism from pathogens present in the intestinal lumen. In this study, we characterized the dynamics of eATP on polarised and non-polarised Caco-2 cells. eATP was quantified by luminometry using the luciferin-luciferase reaction. Results show that non-polarized Caco-2 cells triggered a strong but transient release of intracellular ATP after hypotonic stimuli, leading to low micromolar eATP accumulation. Subsequent eATP hydrolysis mainly determined eATP decay, though this effect could be counterbalanced by eATP synthesis by ecto-kinases kinetically characterized in this study. In polarized Caco-2 cells, eATP showed a faster turnover at the apical vs the basolateral side. To quantify the extent to which different processes contribute to eATP regulation, we created a data-driven mathematical model of the metabolism of extracellular nucleotides. Model simulations showed that eATP recycling by ecto-AK is more efficient a low micromolar eADP concentrations and is favored by the low eADPase activity of Caco-2 cells. Simulations also indicated that a transient eATP increase could be observed upon the addition of non-adenine nucleotides due the high ecto-NDPK activity in these cells. Model parameters showed that ecto-kinases are asymmetrically distributed upon polarization, with the apical side having activity levels generally greater in comparison with the basolateral side or the non-polarized cells. Finally, experiments using human intestinal epithelial cells confirmed the presence of functional ecto-kinases promoting eATP synthesis. The adaptive value of eATP regulation and purinergic signalling in the intestine is discussed. Authors summaryIntestinal epithelial cells play important roles in the absorption of nutrients, secretion of electrolytes and food digestion. When intracellular ATP is released into the intestinal milieu, either at the lumen or the internal side, the resulting extracellular ATP can act as an alert signal to engage cell surface purinergic receptors that activate the immune defence of the organism against pathogens. We worked with Caco-2 and primary human intestinal cell, and our results showed that extracellular ATP regulation is a complex network of reactions that simultaneously consume or generate ATP in whole viable intestinal epithelial cells. In particular, we created a mathematical model, fitted to experimental data, that allowed to quantify the degree to which intracellular ATP release and the activity of a variety of ectoenzymes controlling the concentration of extracellular ATP in a complex way.

biochemistry↗